CN115813980A - Application of pharmaceutical composition in preparation of pseudomonas aeruginosa drug-resistant strain biofilm inhibitor - Google Patents
Application of pharmaceutical composition in preparation of pseudomonas aeruginosa drug-resistant strain biofilm inhibitor Download PDFInfo
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Abstract
The invention discloses an application of a pharmaceutical composition in preparing a pseudomonas aeruginosa biofilm inhibitor, wherein the pseudomonas aeruginosa comprises a pseudomonas aeruginosa standard strain and/or a pseudomonas aeruginosa drug-resistant strain, the pharmaceutical composition comprises a traditional Chinese medicine extract and levofloxacin, and the preparation method of the traditional Chinese medicine extract comprises the following steps: weighing appropriate amount of radix astragali, flos Lonicerae, radix Angelicae sinensis, herba Artemisiae Annuae and rhizoma Polygoni Cuspidati, and extracting with water and precipitating with ethanol to obtain the Chinese medicinal extract. Research shows that compared with the single levofloxacin group, the levofloxacin group combined traditional Chinese medicine extract has obvious reduction in the forming amount of pseudomonas aeruginosa drug-resistant bacteria biomembrane, and the difference has statistical significance, so that the traditional Chinese medicine extract can synergistically enhance the inhibition effect of levofloxacin on the drug-resistant mechanism of pseudomonas aeruginosa biomembrane.
Description
Technical Field
The invention relates to the technical field of medicines, and particularly relates to an application of a pharmaceutical composition in preparation of a pseudomonas aeruginosa drug-resistant strain biofilm inhibitor.
Background
Pseudomonas Aeruginosa (PA) is a non-fermentative opportunistic pathogenic gram-negative bacillus, widely present in the surrounding environment, often causing nosocomial infections. According to statistics, the infection of the pseudomonas aeruginosa accounts for 20 to 30 percent of the acquired infection in hospitals, and the infection tends to increase year by year, thus seriously harming the life health of human beings. With the development of science and technology, people have increasingly studied the drug resistance mechanism of bacteria. Modern researches show that the drug resistance mechanism of pseudomonas aeruginosa is mainly divided into mechanisms of changing the action target of an antibacterial drug, forming a biological membrane, an active efflux system, an active efflux pump system and the like. The biomembrane drug resistance is one of typical adaptive drug resistance mechanisms, and the biomembrane can help PA avoid the elimination of the human immune system and the attack of antibiotics, and has drug resistance to common antibacterial drugs, thereby bringing great challenges to clinical treatment.
Biofilm (BF) is a natural form of bacterial growth that is ubiquitous in the natural environment. The mushroom membrane mainly comprises bacteria and secreted proteins, extracellular polysaccharide, phospholipid, peptidoglycan and other substances, forms a mushroom-shaped membrane structure after maturation, is adhered to the surface of a cavity of a living body or an inanimate substance, has strong adhesion, can be continuously regenerated, and is extremely difficult to remove. Research shows that after bacteria form a biological membrane, the drug resistance to antibiotics can be increased by 10-1000 times, and the harm is great. In addition, the biomembrane is estimated to participate in mediating 65-80% of infections in human bodies, the infected people are mainly mechanical ventilation related patients, burn patients and low-immunity old patients in hospitals, the number of the infected people is large, the treatment cost is high, and great challenges are brought to a social public medical system.
In recent research reports, multiple-drug-resistant, pan-drug-resistant and full-drug-resistant strains are frequently found, and have already formed serious threats to public health safety. With the wide application of antibiotics, the antibacterial activity of a single antibiotic is reduced, and the treatment effect is poor. The treatment scheme of combining multiple antibiotics can ensure the antibacterial activity of the antibiotics under the conventional dosage, and the clinical treatment effect is good. However, the treatment scheme of combining multiple antibiotics cannot radically solve the problem of generation of drug-resistant bacteria, and may provide suitable conditions for generation of the drug-resistant bacteria. In addition, research shows that when multiple antibiotics are in sub-antibacterial concentration, the antibiotics can induce bacterial biofilm drug resistance and promote the generation of virulence factors, and the harm is great. Therefore, the drug administration strategy of searching new drugs for replacing or reducing the combined type of antibiotics, reducing the drug administration dosage of the antibiotics and preventing and treating the bacterial biofilm drug resistance is imperative.
Disclosure of Invention
Based on the above, the invention provides an application of a pharmaceutical composition in preparation of a pseudomonas aeruginosa biofilm inhibitor, wherein the pseudomonas aeruginosa comprises a pseudomonas aeruginosa standard strain and/or a pseudomonas aeruginosa drug-resistant strain, the pharmaceutical composition comprises a traditional Chinese medicine extract and levofloxacin, and the preparation method of the traditional Chinese medicine extract comprises the following steps: weighing an appropriate amount of five traditional Chinese medicine decoction pieces of astragalus, honeysuckle, angelica, sweet wormwood and giant knotweed, and preparing the traditional Chinese medicine extract by a water extraction and alcohol precipitation method.
According to another aspect of the present invention, there is provided a use of a formulation comprising a pharmaceutical composition for the preparation of a pseudomonas aeruginosa biofilm inhibitor, the pseudomonas aeruginosa comprising a pseudomonas aeruginosa standard strain and/or a pseudomonas aeruginosa drug-resistant strain, wherein the pharmaceutical composition comprises a traditional Chinese medicine extract and levofloxacin, wherein the traditional Chinese medicine extract is prepared by a method comprising: weighing appropriate amount of radix astragali, flos Lonicerae, radix Angelicae sinensis, herba Artemisiae Annuae and rhizoma Polygoni Cuspidati, and extracting with water and precipitating with ethanol to obtain the Chinese medicinal extract.
Further, the preparation comprises one or more pharmaceutically acceptable pharmaceutical excipients.
Further, the pharmaceutical adjuvant is selected from one or more of preservatives, wetting agents, humectants, fragrances, surfactants, emulsifiers, thickeners, and lubricants.
Furthermore, the weight ratio of the five traditional Chinese medicine decoction pieces is (8-16): 1-5.
Furthermore, the weight ratio of the five traditional Chinese medicine decoction pieces is (10-14) to (2-4) to (1-3).
Further, the weight ratio of the five traditional Chinese medicine decoction pieces is about 12: about 3: about 2.
Furthermore, the weight ratio of the five traditional Chinese medicine decoction pieces is (11.4-12.6): (2.85-3.15): (1.9-2.1).
Further, the water extraction and alcohol precipitation method comprises the following steps: reflux-extracting for 2-4 times by using 10-12 times of water in sequence, each time for 0.5-2 h, combining the water extract, mixing uniformly, and concentrating to obtain a concentrated solution with crude drug concentration of 0.5-2.0 g/mL; after the concentrated solution is cooled to room temperature, adding a certain volume of ethanol until the alcohol concentration is 50-70%, and refrigerating and standing at 2-8 ℃ for 18-30 h; filtering to obtain supernatant, volatilizing until no alcohol smell, and concentrating to obtain Chinese medicine extract with crude drug concentration of 1.0-3.0 g/mL.
Further, the water extraction and alcohol precipitation method comprises the following steps: sequentially extracting with 12, 10 and 10 times of deionized water under reflux for 3 times (each time for about 1 hr), mixing 3 water extractive solutions, mixing, and concentrating to obtain concentrated solution with crude drug concentration of about 1.0 g/mL; after the concentrated solution is cooled to room temperature, adding a certain volume of ethanol until the alcohol concentration is about 60%, and refrigerating and standing at about 4 ℃ for about 24 hours; filtering to obtain supernatant, volatilizing to remove alcohol smell, and concentrating to obtain Chinese medicinal extract with crude drug concentration of about 2.0 g/mL.
Further, the water extraction and alcohol precipitation method comprises the following steps: reflux-extracting with deionized water of 12, 10 and 10 times in turn for 3 times, each time for 0.95-1.05 h, combining the water extract of 3 times, mixing uniformly, and concentrating to obtain a concentrated solution with crude drug concentration of 0.95-1.05 g/mL; after the concentrated solution is cooled to room temperature, adding ethanol with a certain volume until the alcohol concentration is 57-63%, refrigerating and standing at the temperature of 3.8-4.2 ℃ for 22.8-25.2 h; filtering to obtain supernatant, volatilizing until no alcohol smell, and concentrating to obtain Chinese medicine extract with crude drug concentration of 1.9-2.1 g/mL.
Furthermore, the mass ratio of the traditional Chinese medicine extract to the levofloxacin is (90-200) to 1.
Furthermore, the mass ratio of the traditional Chinese medicine extract to the levofloxacin is (150-195) to 1.
Furthermore, the mass ratio of the traditional Chinese medicine extract to the levofloxacin is (173.85-192.15): 1;
further, the mass ratio of the traditional Chinese medicine extract to the levofloxacin is about 183.3125.
Further, the biofilm inhibitor is an inhibitor that inhibits biofilm formation.
The invention has the beneficial effects that:
compared with the levofloxacin single-use group, the levofloxacin combination of the traditional Chinese medicine extract group has the advantages that the forming amount of pseudomonas aeruginosa drug-resistant bacteria biofilm is obviously reduced, and the difference has statistical significance. Research results suggest that the traditional Chinese medicine extract can synergistically enhance the inhibition effect of levofloxacin on the drug resistance mechanism of pseudomonas aeruginosa biomembranes.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is apparent that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without departing from the scope of the present invention as claimed.
FIG. 1 is a schematic drawing showing the streaking results of a P.aeruginosa activation plate.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
As used in the specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.
Unless defined otherwise, all technical and scientific terms and abbreviations used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this term applies. Although any methods, conditions, materials, or materials similar or equivalent to those disclosed herein can be used in the practice of the present invention, the preferred methods, conditions, materials, or materials are described herein.
The present invention is intended to cover all alternatives, modifications and equivalents, which may be included in the field of the present invention as defined by the appended claims. Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein which can be used in the practice of the present invention. The present invention is in no way limited to the description of methods and materials.
In the present invention, the term "comprising" is synonymous with "including". The terms "comprises," "comprising," "includes," "including," "has," "having," "contains," "containing," or any other variation thereof, as used herein, are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.
As described in the background section, multiple-resistant, pan-resistant, and fully-resistant strains of bacteria are rare and have posed a serious threat to public health and safety. With the wide application of antibiotics, the antibacterial activity of a single antibiotic is reduced, and the treatment effect is poor. The treatment scheme of combining multiple antibiotics can ensure the antibacterial activity of the antibiotics under the conventional dosage, and the clinical treatment effect is good. However, the treatment scheme of combining multiple antibiotics cannot radically solve the problem of generation of drug-resistant bacteria, and may provide suitable conditions for generation of the drug-resistant bacteria. In addition, research shows that when multiple antibiotics are in sub-antibacterial concentration, the antibiotics can induce bacterial biofilm drug resistance and promote the generation of virulence factors, and the harm is great. In order to solve the above problems, the present invention provides a use of a pharmaceutical composition for preparing a pseudomonas aeruginosa biofilm inhibitor, wherein the pseudomonas aeruginosa comprises a pseudomonas aeruginosa standard strain and/or a pseudomonas aeruginosa drug-resistant strain, wherein the pharmaceutical composition comprises a traditional Chinese medicine extract and levofloxacin, and the preparation method of the traditional Chinese medicine extract comprises: weighing appropriate amount of radix astragali, flos Lonicerae, radix Angelicae sinensis, herba Artemisiae Annuae and rhizoma Polygoni Cuspidati, and extracting with water and precipitating with ethanol to obtain the Chinese medicinal extract.
The pseudomonas aeruginosa is a common gram-negative pathogen in nosocomial infection, has high detection rate, low cure rate and complicated drug resistance mechanism, and seriously harms the life health of human beings. Biofilm (BF) is a typical mechanism for the adaptive resistance mechanism of pseudomonas aeruginosa. Once the biofilm has matured, the bacteria will have a greater capacity to adapt to the external environment. Research shows that compared with planktonic bacteria, the resistance of pathogenic bacteria in a biofilm mature state to antibiotics is 10-1000 times higher. Because the biomembrane drug resistance has reversibility, the biomembrane drug resistance can be used for the research of reversing drug resistance of drug-resistant bacteria, and has great research value.
According to another aspect of the present invention, there is provided a use of a formulation comprising a pharmaceutical composition for the preparation of a pseudomonas aeruginosa biofilm inhibitor, the pseudomonas aeruginosa comprising a pseudomonas aeruginosa standard strain and/or a pseudomonas aeruginosa drug-resistant strain, wherein the pharmaceutical composition comprises a traditional Chinese medicine extract and levofloxacin, wherein the traditional Chinese medicine extract is prepared by a method comprising: weighing appropriate amount of radix astragali, flos Lonicerae, radix Angelicae sinensis, herba Artemisiae Annuae and rhizoma Polygoni Cuspidati, and extracting with water and precipitating with ethanol to obtain the Chinese medicinal extract.
In a preferred embodiment, the formulation includes one or more pharmaceutically acceptable pharmaceutical excipients.
In a preferred embodiment, the pharmaceutical excipient is selected from one or more of preservatives, wetting agents, humectants, fragrances, surfactants, emulsifiers, thickeners and lubricants.
In a preferred embodiment, the weight ratio of the five traditional Chinese medicine decoction pieces is (8-16): 1-5: (1-5).
In the present invention, when ratios, equivalents, concentrations, temperatures, times, parts by weight, or other values or parameters are expressed as ranges, preferred ranges, or ranges bounded by a list of upper preferable values and lower preferable values, this is to be understood as specifically disclosing all ranges formed from any pair of any upper range limit or preferred value and any lower range limit or preferred value, regardless of whether ranges are separately disclosed. For example, when the ranges "8 to 16" and "1 to 5" are disclosed, unless otherwise specified, the ranges are intended to include the endpoints thereof and all integers and fractions within the ranges, and the effects of the present invention can be achieved within the ranges disclosed above.
In a preferred embodiment, the weight ratio of the five traditional Chinese medicine decoction pieces is (10-14) to (2-4) to (1-3).
In a preferred embodiment, the weight parts ratio of the five herbal pieces is about 12: about 3: about 2.
In the present invention, "about" refers to a value within a range of ± 5% of a specific value. For example, "about 12" includes ± 5% of 12, i.e. from 11.4 to 12.6;
in a preferred embodiment, the weight ratio of the five traditional Chinese medicine decoction pieces is (11.4-12.6): (2.85-3.15): (1.9-2.1): (2.85-3.15): 1.9-2.1).
In a preferred embodiment, the aqueous extraction and alcohol precipitation method comprises the following steps: reflux-extracting for 2-4 times by using 10-12 times of water in sequence, each time for 0.5-2 h, combining the water extract, mixing uniformly, and concentrating to obtain a concentrated solution with crude drug concentration of 0.5-2.0 g/mL; after the concentrated solution is cooled to room temperature, adding ethanol with a certain volume until the alcohol concentration is 50-70%, and refrigerating and standing for 18-30 h at the temperature of 2-8 ℃; filtering to obtain supernatant, volatilizing until no alcohol smell, and concentrating to obtain Chinese medicine extract with crude drug concentration of 1.0-3.0 g/mL.
In a preferred embodiment, the aqueous extraction and alcohol precipitation method comprises the steps of: sequentially extracting with 12, 10 and 10 times of deionized water under reflux for 3 times (each time for about 1 hr), mixing 3 water extractive solutions, mixing, and concentrating to obtain concentrated solution with crude drug concentration of about 1.0 g/mL; after the concentrated solution is cooled to room temperature, adding a certain volume of ethanol until the alcohol concentration is about 60%, and refrigerating and standing at about 4 ℃ for about 24 hours; filtering to obtain supernatant, volatilizing to remove alcohol smell, and concentrating to obtain Chinese medicinal extract with crude drug concentration of about 2.0 g/mL.
In a preferred embodiment, the aqueous extraction and alcohol precipitation method comprises the following steps: reflux-extracting with deionized water of 12, 10 and 10 times in turn for 3 times, each time for 0.95-1.05 h, combining the water extract of 3 times, mixing uniformly, and concentrating to obtain a concentrated solution with crude drug concentration of 0.95-1.05 g/mL; after the concentrated solution is cooled to room temperature, adding a certain volume of ethanol until the alcohol concentration is 57-63%, refrigerating and standing at 3.8-4.2 ℃ for 22.8-25.2 h; filtering to obtain supernatant, volatilizing until no alcohol smell, and concentrating to obtain Chinese medicine extract with crude drug concentration of 1.9-2.1 g/mL.
In a preferred embodiment, the mass ratio of the traditional Chinese medicine extract to the levofloxacin is (90-200): 1.
In a preferred embodiment, the mass ratio of the traditional Chinese medicine extract to the levofloxacin is (150-195): 1.
In a preferred embodiment, the mass ratio of the traditional Chinese medicine extract to the levofloxacin is (173.85-192.15) 1;
in a preferred embodiment, the mass ratio of the herbal extract to the levofloxacin is about 183.3125.
In a preferred embodiment, the biofilm inhibitor is an inhibitor that retards biofilm formation.
The present invention also provides a method of inhibiting the formation of pseudomonas aeruginosa biofilms, comprising administering an effective amount of the above pharmaceutical composition. The dosage will, of course, vary depending upon the mode of administration, the treatment desired and the condition to be treated.
It should be noted that, in the present application, the embodiments and features of the embodiments may be combined with each other without conflict. The present invention will be described in detail with reference to examples.
The present invention is described in further detail below with reference to specific examples, which are not to be construed as limiting the scope of the invention as claimed herein.
Examples
Laboratory apparatus
Temperature-adjusting electric heating jacket, beijing Zhongxing Weiwei Instrument Co., ltd. (model: ZDHW); electronic balance, sidolisi scientific instruments Beijing, inc. (model: CPA 2250); an electric heating constant temperature air-blast drying oven, shanghai-constant science and technology Co., ltd (model: DHG-9070A); a vertical pressure steam sterilizer, shanghai Boxun industries, inc. (model: YXQ-100A); an electric heating constant temperature water bath, beijing Kewei Yongxing instruments Co., ltd. (model: HH-S6); a circulating water type multipurpose vacuum pump, zhengzhou great wall science and trade company, inc. (model: SHB-III); enzyme-labeled analyzer, beijing Langchen technology Co., ltd (model: DNM-9602); a microbial programmable incubator, rad tai \ [ 21194; class II A2 biosafety cabinet, seimer feishale (suzhou) instruments ltd (model: 1374); vortex mixer, model number XW-80A, manufactured by Linbel instruments, ohio, haimen; micropipettes, eppendorf, germany; a low temperature freezer at-80 ℃ from Thermo company, USA (model: ULT 1386-3-V42); ultracentrifuge, SIGMA corporation, USA (model: sartorius 1-14); water bath nitrogen blowing instrument, beijing Yongwei science and technology Limited (model: CM-24); a gas bath constant temperature oscillator, model ZD-85, from Han mechanical equipments, inc. of Jining, city.
Bacterial strains
Standard pseudomonas aeruginosa ATCC27853 generation strain (accession No. BNCC 337940) provided by the institute of biotechnology, initiatives, beijing; the multiple drug-resistant pseudomonas aeruginosa clinical isolate (sample number 19PXSP 2218) was provided by the university of traditional Chinese medicine, tokyo, dongdu hospital clinical laboratory.
Test reagents and drugs
Tryptone soy broth (TSB, lot # 20200930), nutrient agar (NA, lot # 20200921), M-H agar (MHA, lot # 20210124), agar powder (lot # 20190422) were purchased from Qingdao Hippon Biotech, hippocampus, inc. M-H broth (MHB, batch No.: 1126T 031) was purchased from Beijing Soilebao technologies, inc. Peptone (batch number: 20190102) was purchased from Beijing Bootouda technologies, inc. Levofloxacin standards (CAS #100986-85-4, HPLC ≥ 99%), ciprofloxacin hydrochloride (CAS #93107-08-5, HPLC ≥ 98%), orcinol (3, 5-dihydroxytoluene, AR, lot # S30251), 0.4% ammonium oxalate crystal violet stain (lot # R20710), 2.5% glutaraldehyde stationary (lot # L26O11G 128919), magnesium chloride (AR, lot # S01A10Y 84450), glycerol (AR), and potassium sulfate (AR) were purchased from Shanghai-sourced leaf biotechnology Limited. 95% ethanol, ethyl Acetate (AR) was purchased from Beijing Honghu Co-Ltd. Sodium chloride injection (batch No. 1905052103) was purchased from Shijiazhuang Siyao, inc. Concentrated sulfuric Acid (AR), concentrated hydrochloric Acid (AR) and chloroform (AR) are provided by a dangerous chemical management warehouse of Beijing university of traditional Chinese medicine.
Astragalus root, beijing san and Yao corporation (Productivity: gansu, batch No. 90541102); honeysuckle of Chinese herbal pieces decoction, beijing san and pharmaceutical Co., ltd (Henan, batch No. 93451002); chinese angelica, beijing san and pharmaceutical Co., ltd (Productivity: gansu, batch No. 90280901); chinese medicinal decoction pieces herba Artemisiae Annuae, beijing Sanhe and pharmaceutical Co., ltd (Hebei, production area: 93051001); giant knotweed rhizome, beijing Sanzhou and pharmaceutical Co., ltd (producing area: jiangsu, batch No. 90510501).
Experimental equipment
60mm sterile petri dish, min & science and technology (Beijing); 24-well culture plates, corning-Costar, USA (Cat. No.: 3524); a 96-well U-bottom plate, corning-Costar, USA (Cat: 3799); 96-well culture plates, corning-Costar, USA (Cat. No.: 3599); 50mL sterile centrifuge tubes, corning-Costar, USA (Cat: 430828); 15mL sterile centrifuge tubes, corning-Costar, USA (Cat. No.: 430791); syringe type filters, available from seupitz (beijing) technologies ltd (cat # GL1320 SX).
Related instruments: rat gavage needles, negative pressure vacuum sterile blood collection tubes, sterile cotton balls, medical disposable blood collection needles, 1mL syringes, cotton soft tissues, 1.5mL centrifuge tube boxes, 12mL sterile shake tubes, 5mL sterile shake tubes, tweezers, enamel trays, inoculating rings, aluminum disinfection boxes, syringe filters and the like were purchased from Seipurjen (Beijing) science and technology Limited.
Data processing
SPSS Statistics 21.0 and Origin2021 are adopted for data Statistics and drawing respectively, and the measurement data are adopted
The expression shows that the statistical significance is provided by adopting single-factor analysis of variance when the normal distribution is met and the variance is uniform, adopting an LSD-T method for comparison among groups, adopting single-factor analysis of variance when the variance is nonuniform according to the normal distribution, adopting a Dunnett-T3 method for comparison among groups, adopting nonparametric test for the metering data which are not in accordance with the normal distribution and expressing the difference with P < 0.05.
Example A Observation of Pseudomonas aeruginosa activation
1 method of experiment
1.1 preparation of bacterial suspension
1.1.1 preparation of the culture Medium
Weighing 1.5g of TSB culture medium (components: tryptone, soybean peptone, sodium chloride, dipotassium hydrogen phosphate and glucose, pH value =7.3 +/-0.2) by using an electronic balance according to the proportion of 30.0g/L in the specification, placing the TSB culture medium into a 150mL conical flask, adding 50mL of distilled water to fully dissolve the TSB culture medium, placing a high-temperature-resistant tissue culture sealing film at the opening of the conical flask, tying and sealing a cotton rope, placing the conical flask into a vertical pressure steam sterilizer for autoclaving at 121 ℃ for 15min, naturally cooling to room temperature, shaking uniformly, opening the conical flask beside an alcohol lamp, burning the opening of the conical flask, then sub-packaging the conical flask into 12mL sterile shake bacteria tubes, each tube being 5-6mL, sealing and storing the sealing film in a refrigerator at 4 ℃ for later use.
Weighing 3.3g of NA culture medium (components: peptone, beef extract powder, sodium chloride and agar, pH value =7.3 +/-0.1) by using an electronic balance according to the proportion of 33.0g/L in the specification, placing the NA culture medium into a 250mL conical flask, adding 100mL of distilled water to fully dissolve the NA culture medium, placing a high-temperature-resistant tissue culture sealing film into a conical flask opening, tying and sealing a cotton rope, placing the conical flask opening into a vertical pressure steam sterilizer for autoclaving at 121 ℃ for 15min, naturally cooling the culture medium until the culture medium is not scalded by touch, shaking the culture medium evenly, opening the culture medium beside an alcohol lamp, burning the flask opening, pouring the flask opening backwards into a sterile culture dish with the diameter of 60mm, flattening the culture medium to the thickness of about 5mm, and storing the culture medium in a refrigerator with the temperature of 4 ℃ for later use after the culture medium is naturally cooled and solidified.
1.1.2 preparation of strains
(1) Strain activation: 1 sterile shake tube containing 5-6mL of TSB medium was prepared and placed at room temperature until the temperature rose. Removing the tube body label of the freeze-dried tube filled with the pseudomonas aeruginosa freeze-dried bacteria powder, wiping the tube wall with a wet cotton ball containing 75% alcohol, and then moving the tube wall into a biological safety cabinet. And (5) igniting the alcohol lamp, and performing tube punching within a sterile range of 10cm beside the alcohol lamp. The tube body is divided into two circles at 1/2 position by a small grinding wheel, the tube body is wiped by a wet cotton ball containing 75% alcohol, the top of the freeze-drying tube is placed on the flame of an alcohol lamp for burning, then sterile physiological saline with lower temperature is quickly dripped to crack the tube wall, and broken glass is slightly knocked off by tweezers. Sucking 0.5mL of TSB culture medium, injecting into the freeze-drying tube, gently blowing and beating for several times, beating back the shake tube after fully dissolving, and mixing uniformly. Placing the TSB shake bacteria tube containing bacteria in a thermostat at 37 ℃ for culturing for 16-20h.
(2) Partitioning and scribing: the agar plate is reversely placed in a10 cm sterile range beside an alcohol lamp, the inoculating loop is ignited by the outer flame of the alcohol lamp by the right hand to be red, and the inoculating loop is cooled for 5 to 6 seconds in the range. Taking out the TSB shake bacteria tube containing bacteria with the left hand, taking down the sealing film, opening the shake bacteria tube cover with the right hand little finger and ring finger, and quickly burning the tube opening by the outer flame of the alcohol lamp. A small amount of PA bacterial liquid is picked by the right hand-held inoculating ring, and the culture dish is quickly opened by the left hand to be perpendicular to the desktop as much as possible. The flat plate is divided into 4 areas including A, B, C and D, a plurality of continuous parallel lines are drawn in the Z shape of the area A, the bottom of the dish is quickly buckled above the dish cover, residual bacteria on the inoculating ring are removed by firing, and the separation effect is prevented from being influenced by excessive bacteria. And (3) after the inoculating loop is cooled for 5-6 seconds, rotating the culture dish at an angle of about 75 degrees, rotating the area B to the upper part, carrying the bacteria to the area B by the inoculating loop through the area A, and drawing a plurality of continuous parallel lines in a Z shape to finish secondary parallel drawing. After the third parallel streaking from the B region to the C region and the fourth parallel streaking from the C region to the D region were successively carried out by the same method, the TSA agar plate containing the bacteria was incubated at 37 ℃ for 16 to 20 hours in an incubator, and the growth of PA was observed. Note whether a single colony is separated from the D region, and the appearance characteristics (shape, size, surface, edge, color, transparency and other characters) of the colony are observed.
2 results of the experiment
After culturing for 16-20h at the constant temperature of 37 ℃, the TSB culture medium in the shake culture tubes of the standard pseudomonas aeruginosa strain and the clinical multi-drug-resistant pseudomonas aeruginosa strain is observed to be changed from clarification to turbidity. After the bacteria liquid streaking agar plate is cultured at the constant temperature of 37 ℃ for 16-20h, typical PA bacterial colonies can be seen, the size of the bacterial colonies is different, about 2-4mm, the color is light yellow, the shape is flat, the edge is irregular, the middle is convex, and the texture is wet and viscous. The plates had a ginger smell, and most of the areas were significantly greenish. As shown in fig. 1. The PA strain in the experiment has good reactivation property, and the colony morphology meets the requirements.
Example two effects of combination of extracts of Chinese herbs with levofloxacin on biofilm of drug-resistant bacteria
1 method of experiment
1.1 preparation of bacterial suspension
1.1.1 preparation of the culture Medium
Weighing 12.6g of MHA culture medium (components: casein peptone, beef extract powder, soluble starch and agar, pH value =7.3 +/-0.1) by using an electronic balance according to the proportion of 42.0g/L in the specification, placing the MHA culture medium into a conical flask, adding 300mL of distilled water to fully dissolve the MHA culture medium, placing a high-temperature-resistant tissue culture sealing film into a conical flask opening, tying a cotton rope to seal the opening tightly, placing the conical flask opening into a vertical pressure steam sterilizer to sterilize for 15min at 121 ℃, naturally cooling the MHA culture medium until the MHA culture medium is not scalded by touch, shaking the MHA culture medium evenly, opening the MHA culture medium beside an alcohol lamp, pouring the MHA culture medium into a sterile culture dish with the diameter of 60mm after the MHA culture medium is naturally cooled and solidified, and storing the MHA culture medium in a refrigerator with the temperature of 4 ℃ for later use.
1.1.2 preparation of bacterial liquid
Selecting a single pseudomonas aeruginosa drug-resistant bacterium colony, inoculating the single pseudomonas aeruginosa drug-resistant bacterium colony to an MHA plate by adopting a zone streaking method, and placing the MHA plate in a constant-temperature incubator for culturing for 16-20h at 37 ℃.1 sterile 12mL shake tube was prepared and 3-4mL of saline was injected with a syringe. Burning an inoculating loop by using outer flame of an alcohol lamp until the inoculating loop is red, cooling the inoculating loop within the range of 10cm beside the alcohol lamp for 5-6 seconds, picking a typical PA single colony in a D area of an MHA panel, opening a shake bacteria tube cover by using a right hand little finger and a ring finger, holding the shake bacteria tube by using a left hand, quickly burning a tube opening by using the outer flame of the alcohol lamp, placing the inoculating loop on the tube wall of the shake bacteria tube, grinding and dispersing sticky colonies, taking a sterile 3mL dropper, repeatedly blowing and beating to suspend the PA, performing visual measurement and turbidimetry by using a remel0.5 McLenti tube, and adjusting the concentration of a bacteria solutionTo a concentration of 0.5 McLeod (1.5X 10) 8 CFU/mL), mixed by repeatedly pipetting with a pipette, and 200 μ L was pipetted into a 96-well plate, and absorbance at a wavelength of 600nm was measured (OD 600= 0.11) in 3 duplicate wells. The preparation method of the bacterial liquid with the concentration of 4MCF is the same, and the preparation is finished when the concentration of the bacterial liquid is 0.5MCF after the bacterial liquid is diluted by 8 times. The bacterial suspension was prepared just before each experiment.
1.2 preparation of intervention liquid medicine
1.2.1 preparation of extracts of Chinese herbs
Weighing five traditional Chinese medicine decoction pieces including astragalus, honeysuckle, angelica, sweet wormwood herb and giant knotweed rhizome according to the weight parts (12. Preparing the traditional Chinese medicine extract by adopting a water extraction and alcohol precipitation method: sequentially extracting with 12, 10 and 10 times of deionized water under reflux for 3 times, each for 1 hr, mixing 3 water extractive solutions, placing in 2L beaker, mixing, pouring into evaporating dish, placing in water bath, and concentrating to obtain 1.0g/mL crude drug concentrated solution; after the concentrated solution is cooled to room temperature, adding a certain volume of ethanol until the concentration of the ethanol is 60%, refrigerating and standing for 24h at 4 ℃ (attention is paid to slow and fast stirring in the process of adding the ethanol so as to avoid generating large-particle floccules and influencing the effect of alcohol precipitation); filtering to obtain supernatant, placing in evaporating dish, evaporating in water bath, concentrating to 2.0g/mL concentration, sealing, and storing at-80 deg.C.
1.2.2 preparation of levofloxacin medicinal liquid
And (3) precisely weighing 80mg of levofloxacin standard, sucking 1mL of physiological saline by using a pipette to fully dissolve the levofloxacin standard, further diluting the liquid medicine by using the physiological saline, sterilizing by using a 0.22 mu L microporous filter membrane to prepare 200 mu g/mL of levofloxacin standard solution, sealing and storing at-20 ℃.
1.3 measurement of biofilm formation amount
1.3.1 preparation of the culture Medium
Weighing 7.2g of MHB culture medium (the components are beef extract powder, starch and casein hydrolysate, the pH value is =7.2 +/-0.2) by using an electronic balance according to the proportion of 24.0g/L in the specification, placing the MHB culture medium into a conical flask, adding 300mL of distilled water to fully dissolve the MHB culture medium, placing a high-temperature-resistant tissue culture sealing film into the conical flask opening, knotting cotton ropes to seal the MHB culture sealing film tightly, placing the MHB culture sealing film into a vertical pressure steam sterilizer to sterilize for 15min at 121 ℃, and naturally cooling to room temperature for later use.
1.3.2 preparation of drug-resistant bacteria biofilm intervention liquid medicine
(1) Levofloxacin (LEV) group: 200 mu g/mL of levofloxacin standard solution is diluted into 4.0 and 2.0 mu g/mL levofloxacin liquid medicines by using MHB culture medium, which respectively correspond to the high-concentration levofloxacin liquid medicine and the low-concentration levofloxacin liquid medicine.
(2) The traditional Chinese medicine extract is combined with levofloxacin (LEV + ZY) group: 200 mu g/mL levofloxacin standard solution is diluted into 8.0 and 4.0 mu g/mL levofloxacin liquid medicine by MHB culture medium. Then 2g/mL of the Chinese medicinal extract is diluted into 1466.7 and 733.3 μ g/mL of Chinese medicinal extract liquid medicine by MHB culture medium. Sequentially mixing the levofloxacin group and the Chinese medicinal extract liquid from high concentration to low concentration in pairs at equal volumes to obtain 2 combined liquid medicines respectively corresponding to the high-concentration combined liquid medicine and the low-concentration combined liquid medicine.
1.3.3 measurement of biofilm by Crystal Violet staining method
(1) Inoculation of bacteria: respectively inoculating 10 mu L of 0.5MCF pseudomonas aeruginosa liquid bacteria into 1mL of levofloxacin group liquid medicine and traditional Chinese medicine extract combined levofloxacin group liquid medicine, blowing, uniformly mixing, sucking 200 mu L of bacteria-containing solution, adding into a U-bottom 96-pore plate, arranging a positive control pore and a negative control pore, arranging 6 compound pores in each group, and repeating the experiment for 3 times.
(2) Establishment of a biological membrane: the 96-well plate in (1) above was placed in an incubator at 37 ℃ for culture. The culture time of the pseudomonas aeruginosa standard bacteria is 24 hours, and the culture time of the pseudomonas aeruginosa drug-resistant bacteria is 48 hours.
(3) Crystal violet dyeing: after the culture in the step (2) is finished, taking out the 96-well plate, carefully pouring out the bacterial liquid in the hole, sucking 200 mu L of physiological saline by using a discharge gun, adding the physiological saline into the hole, gently and slowly adding the liquid, and washing for 2 times in total, so that the floating bacteria are removed. Laying 3 layers of cotton soft towel on a 96-well plate, reversely buckling and sucking off residual liquid in the well, adding 200 mu L of 2.5% glutaraldehyde for fixation for 20min (keeping out of the sun), and carefully pouring off the liquid after fixation is completed. Laying 3 layers of cotton soft towel on the 96-hole plate, and reversely buckling to suck the residual liquid in the hole. The discharging gun sucks 200 mu L of physiological saline and adds the physiological saline into the hole, the liquid adding action is gentle and slow, and the washing is carried out for 2 times. Laying 3 layers of cotton soft towel on the 96-hole plate, and reversely buckling to suck the liquid in the hole. 200 μ L of 0.4% crystal violet staining solution was pipetted into the wells and stained for 15min (protected from light). After staining was complete the liquid was carefully poured off. And (4) sucking 200 mu L of physiological saline by a line gun to clean the 96-well plate for 3-4 times, and cleaning the visible impurities. Laying 3 layers of cotton soft towels on a 96-pore plate, reversely buckling and absorbing liquid in the pores, placing and drying in the dark at room temperature until no obvious liquid exists in the pores, absorbing 200 mu L of 95% ethanol by a discharge gun, adding the ethanol into the pores, decoloring and dissolving crystal violet in a biological membrane for 10 minutes, and detecting the absorbance value at 595nm by using an enzyme labeling instrument.
2 results of the experiment
The results of the effect of the combination of the traditional Chinese medicine extract and levofloxacin on the biofilm of the pseudomonas aeruginosa drug-resistant strain are shown in table 1. The test result shows that compared with a blank control group (namely that no medicine group is added into the drug-resistant strain), the biofilm formation amount of the pseudomonas aeruginosa drug-resistant strain of the single levofloxacin group is obviously reduced (P is less than 0.01); compared with a blank control group, the traditional Chinese medicine extract combined with levofloxacin has obviously reduced biofilm formation amount (P is less than 0.01) in each concentration group. Compared with the single levofloxacin group, the traditional Chinese medicine extract combined levofloxacin group has obviously reduced biofilm formation amount (P is less than 0.01) and shows obvious synergistic effect.
TABLE 1 influence of combination of Chinese medicinal extracts with levofloxacin on biofilm formation of PA-resistant strains (OD 595) ((
n＝5)
Note: * : vs blank P < 0.05; ** : vs blank P < 0.01; # ：vs LEV P＜0.05； ## ：vs LEV P＜0.01。
the foregoing is merely an example of the present invention and common general knowledge of known specific structures and features in the schemes is not described herein in detail. It should be noted that, for those skilled in the art, without departing from the present invention, several changes and modifications can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicability of the patent. The scope of the claims of the present application shall be determined by the contents of the claims, and the description of the embodiments and the like in the specification shall be used to explain the contents of the claims.
The embodiments of the present invention have been described in detail, and the principles and embodiments of the present invention are explained herein using specific examples, which are provided only to help understand the method and the core idea of the present invention. Meanwhile, those skilled in the art should also be able to make modifications or variations to the embodiments and applications of the present invention based on the idea of the present invention. In view of the above, the present disclosure should not be construed as limiting the invention.
Claims (10)
1. Use of a pharmaceutical composition for preparing a pseudomonas aeruginosa biofilm inhibitor, wherein the pseudomonas aeruginosa comprises a pseudomonas aeruginosa standard strain and/or a pseudomonas aeruginosa drug-resistant strain, wherein the pharmaceutical composition comprises a traditional Chinese medicine extract and levofloxacin, wherein the preparation method of the traditional Chinese medicine extract comprises the following steps: weighing appropriate amount of five traditional Chinese medicine decoction pieces of astragalus, honeysuckle, angelica, sweet wormwood and giant knotweed, and preparing the traditional Chinese medicine extract by a water extraction and alcohol precipitation method.
2. Use of a formulation comprising a pharmaceutical composition for the preparation of a pseudomonas aeruginosa biofilm inhibitor, wherein said pseudomonas aeruginosa comprises a pseudomonas aeruginosa standard strain and/or a pseudomonas aeruginosa drug resistant strain, wherein said pharmaceutical composition comprises a traditional Chinese medicine extract and levofloxacin, wherein said traditional Chinese medicine extract is prepared by a method comprising: weighing appropriate amount of five traditional Chinese medicine decoction pieces of radix astragali, flos Lonicerae, radix Angelicae sinensis, herba Artemisiae Annuae and rhizoma Polygoni Cuspidati respectively, and preparing the traditional Chinese medicine extract by water extraction and alcohol precipitation;
preferably, the formulation comprises one or more pharmaceutically acceptable pharmaceutical excipients;
more preferably, the pharmaceutical excipients are selected from one or more of preservatives, wetting agents, humectants, fragrances, surfactants, emulsifiers, thickeners and lubricants.
3. The use as claimed in claim 1 or 2, wherein the weight ratio of the five Chinese medicinal decoction pieces is (8-16): (1-5): 1-5;
preferably, the weight ratio of the five traditional Chinese medicine decoction pieces is (10-14): 2-4): 1-3.
4. The use of claim 3, wherein the ratio of the five herbal pieces of traditional Chinese medicine is about 12: about 3: about 2;
preferably, the weight ratio of the five traditional Chinese medicine decoction pieces is (11.4-12.6) to (2.85-3.15) to (1.9-2.1).
5. Use according to claim 1 or 2, characterized in that said aqueous extraction-alcohol precipitation method comprises the following steps: reflux-extracting for 2-4 times by using 10-12 times of water in sequence, each time for 0.5-2 h, combining the water extract, mixing uniformly, and concentrating to obtain a concentrated solution with crude drug concentration of 0.5-2.0 g/mL; after the concentrated solution is cooled to room temperature, adding a certain volume of ethanol until the alcohol concentration is 50-70%, and refrigerating and standing at 2-8 ℃ for 18-30 h; filtering to obtain supernatant, volatilizing until no alcohol smell, and concentrating to obtain the Chinese medicine extract with crude drug concentration of 1.0-3.0 g/mL.
6. The use according to claim 5, wherein the water extraction and alcohol precipitation method comprises the following steps: sequentially extracting with 12, 10 and 10 times of deionized water under reflux for 3 times (each time for about 1 hr), mixing 3 water extractive solutions, mixing, and concentrating to obtain concentrated solution with crude drug concentration of about 1.0 g/mL; after the concentrated solution is cooled to room temperature, adding a certain volume of ethanol until the alcohol concentration is about 60%, and refrigerating and standing at about 4 ℃ for about 24 hours; filtering to obtain supernatant, volatilizing until no alcohol smell, and concentrating to obtain Chinese medicinal extract with crude drug concentration of about 2.0 g/mL;
preferably, the water extraction and alcohol precipitation method comprises the following steps: reflux-extracting with deionized water of 12, 10 and 10 times in turn for 3 times, each time for 0.95-1.05 h, combining the water extract of 3 times, mixing uniformly, and concentrating to obtain a concentrated solution with crude drug concentration of 0.95-1.05 g/mL; after the concentrated solution is cooled to room temperature, adding ethanol with a certain volume until the alcohol concentration is 57-63%, refrigerating and standing at the temperature of 3.8-4.2 ℃ for 22.8-25.2 h; filtering to obtain supernatant, volatilizing until no alcohol smell, and concentrating to obtain the Chinese medicine extract with crude drug concentration of 1.9-2.1 g/mL.
7. The use according to claim 1 or 2, characterized in that the mass ratio of the traditional Chinese medicine extract to the levofloxacin is (90-200): 1.
8. The use of claim 7, wherein the mass ratio of the traditional Chinese medicine extract to the levofloxacin is (150-195): 1.
9. The use of claim 8, wherein the mass ratio of the traditional Chinese medicine extract to the levofloxacin is (173.85-192.15): 1;
preferably, the mass ratio of the traditional Chinese medicine extract to the levofloxacin is about 183.3125.
10. Use according to any one of claims 1 to 9, wherein the biofilm inhibitor is an inhibitor that retards biofilm formation.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102091134A (en) * | 2011-01-12 | 2011-06-15 | 刘清泉 | Traditional Chinese medicinal composition for resisting multi-medicine tolerance of antibiotics |
| CN106619740A (en) * | 2017-01-03 | 2017-05-10 | 首都医科大学附属北京中医医院 | Model for inducing drug resistance in PA (pseudomonas aeruginosa) animal as well as construction method and application of model |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102091134A (en) * | 2011-01-12 | 2011-06-15 | 刘清泉 | Traditional Chinese medicinal composition for resisting multi-medicine tolerance of antibiotics |
| CN106619740A (en) * | 2017-01-03 | 2017-05-10 | 首都医科大学附属北京中医医院 | Model for inducing drug resistance in PA (pseudomonas aeruginosa) animal as well as construction method and application of model |
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| Title |
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| 石航等: "多指标优选芪归银方的水提醇沉工艺", 《中华中医药学刊》, vol. 33, no. 4, pages 813 - 816 * |
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