CN103720768A - New use of tanreqing in preparation of product for destroying bacterial biofilm - Google Patents
New use of tanreqing in preparation of product for destroying bacterial biofilm Download PDFInfo
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Abstract
The invention discloses new use of tanreqing in preparation of a product for destroying a bacterial biofilm, and provides an application of the tanreqing in preparation of a product for degrading the bacterial biofilm. The invention also discloses the application of the tanreqing in preparation of the product for destroying the bacterial biofilm. The invention also discloses an application of the tanreqing in preparation of a product for inhibiting formation of the bacterial biofilm. The invention finds that the tanreqing not only can inhibit formation of the bacterial biofilm, but also can degrade a mature bacterial biofilm. The tanreqing has important value on public health.
Description
Technical field
The present invention relates to the expectorant heat new purposes of one in the product of preparation destruction bacterial biof iotalm clearly.
Background technology
Bacterial biof iotalm refers to by being attached to the bacterial cell of inertia or active solid object surface and being wrapped in the structural bacterial community that the hydrability substrate of antibacterial forms.Bacterial biof iotalm is extensively present on moisture and moist various surfaces, comprise water supply pipe, industrial pipeline, ventilation installation, medical apparatus and instruments be the human tissue organ under pathological state even, according to expert, estimates that nearly all antibacterial can form biomembrane under certain conditions.In biomembrane the metabolic activity of antibacterial except can corrosion pipeline and metal surface, more can cause animals and plants and human diseases to occur.Because antibacterial has the Drug resistance that exceeds 1,100 times than migration state at biomembrane state, make biomembrane in clinical intractable chronic infection, the serious threat human health of more easily causing.
Tanreqin injection is national Chinese medicine two kind new medicines of being produced, had exclusive intellectual property by Shanghai Kaibao Pharmaceutical Co., Ltd, by scientific composition such as Radix Scutellariae, Fel Ursi powder, Cornu Naemorhedi, Flos Lonicerae, Fructus Forsythiaes, in strict accordance with Chinese medicine fingerprint, produce, have heat-clearing and toxic substances removing, reduce phlegm, spasmolytic, press down the effects such as mattress, antiviral, antipyretic, immunomodulating.The clinical treatment that mainly can be used for the multiple expectorant diseases such as pneumonia, bronchitis, hepatitis, viral encephalitis, infectious monocytosis.
Summary of the invention
The object of this invention is to provide the expectorant heat new purposes of one in the product of preparation destruction bacterial biof iotalm clearly.
The invention provides expectorant heat clear in the application of preparing in the biomembranous product of bacterium for degrading.Described antibacterial can be staphylococcus aureus, specifically can be
number is 25923 staphylococcus aureus.Described expectorant heat can be clearly the dried frozen aquatic products of tanreqin injection or tanreqin injection.Described tanreqin injection specifically can be the tanreqin injection that Shanghai Kaibao Pharmaceutical Co., Ltd produces.
The present invention also protects the application in the product of preparation destruction bacterial biof iotalm clearly of expectorant heat.Described antibacterial can be staphylococcus aureus, specifically can be
number is 25923 staphylococcus aureus.Described expectorant heat can be clearly the dried frozen aquatic products of tanreqin injection or tanreqin injection.Described tanreqin injection specifically can be the tanreqin injection that Shanghai Kaibao Pharmaceutical Co., Ltd produces.
The present invention also protects the clear application in the product of preparing anti-bacteria biofilm formation of expectorant heat.Described antibacterial can be staphylococcus aureus, specifically can be
number is 25923 staphylococcus aureus.Described expectorant heat can be clearly the dried frozen aquatic products of tanreqin injection or tanreqin injection.Described tanreqin injection specifically can be the tanreqin injection that Shanghai Kaibao Pharmaceutical Co., Ltd produces.
The present invention also protects the biomembranous product of a kind of bacterium for degrading, and its active component is that expectorant heat is clear.Described antibacterial can be staphylococcus aureus, specifically can be
number is 25923 staphylococcus aureus.Described expectorant heat can be clearly the dried frozen aquatic products of tanreqin injection or tanreqin injection.Described tanreqin injection specifically can be the tanreqin injection that Shanghai Kaibao Pharmaceutical Co., Ltd produces.
The present invention also protects a kind of product that destroys bacterial biof iotalm, and its active component is that expectorant heat is clear.Described antibacterial can be staphylococcus aureus, specifically can be
number is 25923 staphylococcus aureus.Described expectorant heat can be clearly the dried frozen aquatic products of tanreqin injection or tanreqin injection.Described tanreqin injection specifically can be the tanreqin injection that Shanghai Kaibao Pharmaceutical Co., Ltd produces.
The present invention also protects a kind of product of anti-bacteria biofilm formation, and its active component is that expectorant heat is clear.Described antibacterial can be staphylococcus aureus, specifically can be
number is 25923 staphylococcus aureus.Described expectorant heat can be clearly the dried frozen aquatic products of tanreqin injection or tanreqin injection.Described tanreqin injection specifically can be the tanreqin injection that Shanghai Kaibao Pharmaceutical Co., Ltd produces.
The present invention, take staphylococcus aureus as example, utilizes Microdilution plate method, laser co-focusing, Bioflux shearing force model etc. to study the clear impact on the film formed different times of bacterium living beings, different conditions of expectorant heat.From Microdilution plate method experimental result: expectorant heat is clear, penicillin sodium all has impact to the forming process of bacterial biof iotalm, all can the biomembranous biological total amount of anti-bacteria, and effectively reduce the number of viable in bacterial biof iotalm; When after bacterial biof iotalm maturation, only have the expectorant heat clearly can the biomembranous biological total amount of anti-bacteria, and effectively reduce the number of viable in bacterial biof iotalm, penicillin sodium loses above-mentioned effect.Based on above-mentioned discovery, inventor infers that expectorant heat destroyed clearly bacterium living beings membrane structure, in order to verify this conjecture, inventor by detect through the antibiotic infiltration capacity of the clear filter membrane after treatment of expectorant heat (having ripe bacterial biof iotalm on filter membrane) number, indirectly reflect that expectorant heat is clearly to bacterial biof iotalm structural damage, experimental result shows, increase, and matched group and penicillin sodium processed group can't detect antibiotic infiltration through the antibiotic infiltration capacity of the clear filter membrane after treatment of expectorant heat.For the clear impact on bacterial biof iotalm of observation expectorant heat directly perceived, biomembrane is carried out after viable bacteria (syto-9), dead bacterium (PI) and antibacterial epimatrix (matrix) three marks with fluorescent dye, pass through confocal laser scanning microscope, penicillin sodium (positive control), expectorant heat have remarkable result for the forming process of bacterial biof iotalm clearly, only have expectorant heat clearly ripe bacterial biof iotalm to be produced effect, this is consistent with the result of microwell plate.For the environment of living in of biomembrane in analogue body, inventor has adopted Bioflux shearing force model.From bioflux result: apply penicillin sodium to bacterial biof iotalm, its state and electron density and volume are all without obviously changing; After applying the clear 30min of expectorant heat to bacterial biof iotalm, occur cavity, during 1h, biomembrane starts to peel off with pipeline, to 5h biomembrane, becomes loose, and during 10h, biomembrane dissipates, cracks; This result is consistent with the result of microwell plate result and confocal laser scanning microscope, has all illustrated that expectorant heat has destruction to ripe bacterial biof iotalm clearly, and penicillin sodium to ripe bacterial biof iotalm without effect.
After biomembrane maturation, substrate is wrapped in antibacterial outer surface, forms very orderly structure, and therefore ripe bacterial biof iotalm has very strong drug resistance, so find a kind of medicine that can degrade ripe bacterial biof iotalm to seem very necessary.The present invention finds, expectorant heat clearly can not only the biomembranous formation of anti-bacteria, the ripe bacterial biof iotalm of can also degrading.The present invention has great value for publilc health cause.
Accompanying drawing explanation
Fig. 1 is the result of the step 1 of embodiment 1.
Fig. 2 is the result of the step 2 of embodiment 1.
Fig. 3 is the result (kanamycin sulfate) of the step 3 of embodiment 1.
Fig. 4 is the result (Lyphocin (Fujisawa)) of the step 4 of embodiment 1.
Fig. 5 is the result of the step 1 of embodiment 2.
Fig. 6 is the result of the step 2 of embodiment 2.
Fig. 7 is the result of the step 1 of embodiment 3.
Fig. 8 is the result of the step 2 of embodiment 3.
Fig. 9 is the result of embodiment 4.
The specific embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is conventional method.Test material used in following embodiment, if no special instructions, is and purchases available from routine biochemistry reagent shop.Quantitative test in following examples, all arranges and repeats experiment, results averaged for three times.LIVE/
bacLight Bacterial Viability test kit: U.S. Molecular Probes company).Laser confocal microscope: OLYMPUS, FV1000.Numerical control shear flow living cells automatic analysis platform: BioFlux200systerm.Staphylococcus aureus (Staphylococcus aureus):
number is 25923.
LB culture medium: get tryptone 10g(Britain OXOID company), yeast extract 5g(Britain OXOID company), the Qing Shengda of NaCl10g(Beijing Chemical Engineering Technology company limited), be dissolved in deionized water and be settled to 1L with deionized water.Basic culture solution is the LB culture medium containing 0.25g/100mL glucose (Chemical Reagent Co., Ltd., Sinopharm Group).LB solid medium is the LB culture medium containing 2g/100mL agar.
Tanreqin injection (Shanghai Kaibao Pharmaceutical Co., Ltd): for by Radix Scutellariae, Fel Ursi powder, Cornu Naemorhedi, Flos Lonicerae, Fructus Forsythiae Chinese medicine of the five flavours extract, the accurate word Z20030054 of traditional Chinese medicines; During use, tanreqin injection lyophilization is obtained to dry powder; In embodiment, dry powder is dissolved in to basic culture solution, the dry powder micrograms of the clear concentration of expectorant heat to contain in every milliliter of culture fluid.
Penicillin sodium injection, claims again benzylpenicillin sodium for injection: Huabei Pharmaceutic Co., Ltd, lot number X1108105; During use, the lyophilization of penicillin sodium injection is obtained to dry powder; In embodiment, dry powder is dissolved in to basic culture solution, the dry powder micrograms of the concentration of penicillin sodium to contain in every milliliter of culture fluid.
Kanamycin sulfate injection liquid, claims again kanamycin sulfate, purchased from Beijing DingGuo ChangSheng Biology Technology Co., Ltd, and catalog number DH1774; During use, kanamycin sulfate injection liquid lyophilization is obtained to dry powder; In embodiment, dry powder is dissolved in to basic culture solution, the dry powder micrograms of the concentration of kanamycin sulfate to contain in every milliliter of culture fluid.
Lyphocin (Fujisawa) injection, claims again vancomycin hydrochloride for injection, purchased from Eli Lilly Japan K.K, Seishin Laboratories, catalog number C008056; During use, the lyophilization of Lyphocin (Fujisawa) injection is obtained to dry powder; In embodiment, dry powder is dissolved in to basic culture solution, the dry powder micrograms of the concentration of Lyphocin (Fujisawa) to contain in every milliliter of culture fluid.
For observing the clear impact on bacterial biof iotalm of expectorant heat, the present embodiment has been chosen respectively bacterial biof iotalm and has been formed early stage and bacterial biof iotalm formation two stages of later stage, observes impact and the impact on viable count in bacterial biof iotalm clearly of expectorant heat on the biological total amount of bacterial biof iotalm (biological total amount comprises viable bacteria, dead bacterium and extracellular matrix) clearly of expectorant heat.
One, Microdilution plate method detects expectorant heat clearly on the film formed impact of bacterium living beings
1, packet transaction (every group arranges 5 reprocessings)
First group to the tenth group: staphylococcus aureus is seeded to culture fluid (culture fluid of first group to the tenth group is followed successively by: containing 16500 μ g/ml, 8250 μ g/ml, 4125 μ g/ml, 2063 μ g/ml, 1032 μ g/ml, 516 μ g/ml, 258 μ g/ml, 129 μ g/ml, 65 μ g/ml or the clear basic culture solution of 33 μ g/ml expectorant heat), makes initial OD
600nmvalue be 0.05,37 ℃ standing 24 hours;
The 11 group to the 20 group: staphylococcus aureus is seeded to culture fluid (the 11 group is followed successively by the culture fluid of the 20 group: containing the basic culture solution of 20 μ g/ml, 10 μ g/ml, 5 μ g/ml, 2.5 μ g/ml, 1.25 μ g/ml, 0.625 μ g/ml, 0.31 μ g/ml, 0.15 μ g/ml, 0.075 μ g/ml or 0.038 μ g/ml penicillin sodium), makes initial OD
600nmvalue be 0.05,37 ℃ standing 24 hours;
The 21 group (negative control): staphylococcus aureus is seeded to basic culture solution, makes initial OD
600nmvalue be 0.05,37 ℃ standing 24 hours;
Experiment is carried out in 96 orifice plates.
2, get 96 orifice plates of completing steps 1, with 0.9% normal saline cleaning 2 times (antibacterial adhering to remove Kong Zhongwei).
3, get 96 orifice plates of completing steps 2, adopt crystal violet staining assay to detect the biological total amount in every hole, the results are shown in Figure 1A(*P<0.05).
4, get 96 orifice plates of completing steps 2, adopt XTT staining to detect the viable bacteria amount in every hole, the results are shown in Figure 1B(*P<0.05).
The result of Fig. 1 shows: at bacterial biof iotalm formation stages, the cleer and peaceful penicillin sodium of expectorant heat all can reduce its biological total amount, and the clear useful effect concentration of expectorant heat is 1032-16500 μ g/ml, and the useful effect concentration of penicillin sodium is 0.038-20 μ g/ml; At bacterial biof iotalm formation stages, the cleer and peaceful penicillin sodium of expectorant heat all can reduce its viable bacteria amount, and the useful effect concentration of penicillin sodium is 0.038-20 μ g/ml, and the clear useful effect concentration of expectorant heat is 129-16500 μ g/ml.In each group, the result of 5 reprocessings is consistent.
Two, Microdilution plate method detects the clear impact on ripe bacterial biof iotalm of expectorant heat
1, packet transaction (every group arranges 5 reprocessings)
First group to the tenth group: staphylococcus aureus is seeded to basic culture solution, makes initial OD
600nmvalue is 0.05,37 ℃ standing 24 hours, reject supernatant, add culture fluid (culture fluid of first group to the tenth group is followed successively by: containing 16500 μ g/ml, 8250 μ g/ml, 4125 μ g/ml, 2063 μ g/ml, 1032 μ g/ml, 516 μ g/ml, 258 μ g/ml, 129 μ g/ml, 65 μ g/ml or the clear basic culture solution of 33 μ g/ml expectorant heat), 37 ℃ standing 24 hours;
The 11 group to the 20 group: staphylococcus aureus is seeded to basic culture solution, makes initial OD
600nmvalue is 0.05,37 ℃ standing 24 hours, reject supernatant, add culture fluid (the 11 group is followed successively by the culture fluid of the 20 group: containing the basic culture solution of 20 μ g/ml, 10 μ g/ml, 5 μ g/ml, 2.5 μ g/ml, 1.25 μ g/ml, 0.625 μ g/ml, 0.31 μ g/ml, 0.15 μ g/ml, 0.075 μ g/ml or 0.038 μ g/ml penicillin sodium), 37 ℃ standing 24 hours;
The 21 group (negative control): staphylococcus aureus is seeded to basic culture solution, makes initial OD
600nmvalue be 0.05,37 ℃ standing 24 hours, reject supernatant, adds basic culture solution, 37 ℃ standing 24 hours;
Experiment is carried out in 96 orifice plates.
2, with 2 of step 1.
3, with 3 of step 1, the results are shown in Figure 2A(*P<0.05).
4, with 4 of step 1, the results are shown in Figure 2B(*P<0.05).
The result of Fig. 2 shows: for ripe bacterial biof iotalm, penicillin sodium is to its biological total amount without influence, and expectorant heat can reduce clearly its biological total amount, and the clear useful effect concentration of expectorant heat is 33-1032 μ g/ml; For ripe bacterial biof iotalm, except 20 μ g/ml concentration, penicillin sodium is to its viable bacteria amount without influence, and expectorant heat can reduce clearly its viable bacteria amount, and valid density is 65-16500 μ g/ml.In each group, the result of 5 reprocessings is consistent.
The result of the present embodiment shows, penicillin sodium only has and suppresses or Degradation the forming process of bacterial biof iotalm, and expectorant heat is clear not only to be had inhibition or Degradation, ripe bacterial biof iotalm is also had and suppressed or Degradation the forming process of bacterial biof iotalm.
Three, the impact of other medicines on ripe bacterial biof iotalm
Replace penicillin sodium to carry out the experiment of above-mentioned steps two with kanamycin sulfate, biological total amount the results are shown in Figure 3A(***P<0.05), viable bacteria amount the results are shown in Figure 3B(***P<0.05).Replace penicillin sodium to carry out the experiment of above-mentioned steps two with Lyphocin (Fujisawa), biological total amount the results are shown in Figure 4A(***P<0.05), viable bacteria amount the results are shown in Figure 4B(***P<0.05).Except the kanamycin sulfate of 20 μ g/ml has impact the viable bacteria amount of antibacterial, kanamycin sulfate and Lyphocin (Fujisawa) total biomass and the viable bacteria amount to ripe bacterial biof iotalm all do not make significant difference.
One, under different action time, study carefully the clear effect to biofilm structure of expectorant heat
1, get in 6 orifice plates, every hole adds 4ml OD
600nm=0.05 staphylococcus aureus bacterium liquid (bacterium liquid obtains with the resuspended staphylococcus aureus thalline of basic culture solution), then the filter membrane of a 15 × 15mm is put in every hole, cultivates 48h for 37 ℃, to form ripe bacterial biof iotalm at filter membrane upper surface.
2, packet transaction
First group: after completing steps 1, filter membrane is moved to 37 ℃ of standing 24h in the basic culture solution clear containing the expectorant heat of variable concentrations (being respectively containing 16500 μ g/ml, 8250 μ g/ml, 4125 μ g/ml or the clear basic culture solution of 2062 μ g/ml expectorant heat);
Second group: after completing steps 1, filter membrane is moved to 37 ℃ of standing 24h in the basic culture solution (being respectively the basic culture solution containing 20 μ g/ml, 10 μ g/ml, 5 μ g/ml or 2.5 μ g/ml penicillin sodiums) containing the penicillin sodium of variable concentrations;
The 3rd group (negative control): after completing steps 1, filter membrane is moved to 37 ℃ of standing 24h in basic culture solution.
3, after completing steps 2, filter membrane is positioned over containing on the LB solid medium flat board of 20 μ g/ml penicillin sodiums, on filter membrane, cover the aseptic filter membrane of a 15 new × 15mm, form bacterial biof iotalm sandwich, on new aseptic filter membrane, place again the filter paper that a diameter is 6mm, on filter paper, drip 5 μ l basic culture solution moistenings, 37 ℃ of standing 2h, 4h or 6h.
4, after completing steps 3, get filter paper and be positioned on the LB solid medium flat board that is coated with staphylococcus aureus, cultivating 18-24h for 37 ℃, observing inhibition zone.
Fig. 5 is shown in by the photo of cultivating in step 4 after 24 hours.First group is carried out that step 4 is rear all can observe inhibition zone, and second group and the 3rd group is carried out the rear inhibition zone of all can not observing of step 4.
Two, drug sensitive experiment
Filter paper is positioned on the LB solid medium culture medium flat plate that is coated with staphylococcus aureus, drips 5 μ l above containing the basic culture solution of penicillin sodium or drip 5 μ l containing the clear basic culture solution of expectorant heat, cultivate 18-24h for 37 ℃, observe inhibition zone.Fig. 6 is shown in by the photo of cultivating after 24 hours.Can find out, expectorant heat itself there is no inhibition zone clearly, and penicillin sodium inhibition zone is larger.
The result of comprehensive step 1 and step 2, conclusion is as follows: step 12 in, first group is the clear processed group of expectorant heat, expectorant heat has been destroyed clearly bacterium living beings membrane structure, penicillin sodium in step 3 can and be penetrated in filter paper by bacterial biof iotalm, thereby make filter paper there is bactericidal action; Step 12 in, second group is penicillin sodium processed group, penicillin sodium can not destroy bacterium living beings membrane structure, thereby the penicillin sodium in step 3 cannot be penetrated in filter paper by bacterial biof iotalm, filter paper does not have bactericidal action.Result shows, for ripe bacterial biof iotalm, penicillin sodium does not have and suppresses or Degradation, and expectorant heat has clearly significant inhibition or Degradation.
Embodiment 3, by the clear impact on bacterial biof iotalm of confocal laser scanning microscope expectorant heat
One, on the film formed impact of bacterium living beings
First group (the clear group of expectorant heat): in little glass dish, add containing the clear basic culture solution of 2063 μ g/ml expectorant heat, then inoculate staphylococcus aureus, obtain 2mL initial OD
600nmvalue is 0.05 system, 37 ℃ standing 24 hours, supernatant discarded, with twice (remove and do not adhere to antibacterial) of 0.9% normal saline cleaning, then carry out matrix dyeing (extracellular matrix dyeing), PI dyeing (dead antibacterial is dyeed) and Syto9 dyeing (antibacterial is dyeed to living), on laser confocal microscope, observe (PI, Syto9, the required excitation wavelength of matrix dyestuff are followed successively by 488nm, 559nm and 559nm), utilize imaris software to carry out image and data analysis, the results are shown in Figure 7.
Second group (penicillin sodium group): in little glass dish, add the basic culture solution containing 20 μ g/ml penicillin sodiums, then inoculate staphylococcus aureus, obtain 2mL initial OD
600nmvalue is 0.05 system, 37 ℃ standing 24 hours, supernatant discarded, with twice (remove and do not adhere to antibacterial) of 0.9% normal saline cleaning, then carry out matrix dyeing (extracellular matrix dyeing), PI dyeing (dead antibacterial is dyeed) and Syto9 dye (to or antibacterial dye), on laser confocal microscope, observe (PI, Syto9, the required excitation wavelength of matrix dyestuff are followed successively by 488nm, 559nm and 559nm), utilize imaris software to carry out image and data analysis, the results are shown in Figure 7.
The 3rd group (matched group): in little glass dish, add basic culture solution, then inoculate staphylococcus aureus, obtain 2mL initial OD
600nmvalue is 0.05 system, 37 ℃ standing 24 hours, supernatant discarded, with twice (remove and do not adhere to antibacterial) of 0.9% normal saline cleaning, then carry out matrix dyeing (extracellular matrix dyeing), PI dyeing (dead antibacterial is dyeed) and Syto9 dye (to or antibacterial dye), on laser confocal microscope, observe (PI, Syto9, the required excitation wavelength of matrix dyestuff are followed successively by 488nm, 559nm and 559nm), utilize imaris software to carry out image and data analysis, the results are shown in Figure 7.
By Fig. 7, can be observed, at bacterial biof iotalm formation stages, the bactericidal effect that penicillin sodium and expectorant are hot clear is all fine, and the amount of viable bacteria, dead bacterium and extracellular matrix all greatly reduces, bacterial biof iotalm attenuation, smaller volume, and this is consistent with the testing result of microwell plate.Every group arranges 5 reprocessings.In each group, the result of 5 reprocessings is consistent.
Two, the impact on ripe bacterial biof iotalm
First group (the clear group of expectorant heat): in little glass dish, add basic culture solution, then inoculate staphylococcus aureus, obtain 2mL initial OD
600nmvalue is 0.05 system, and 37 ℃ standing 24 hours; Reject supernatant, adds containing the clear basic culture solution of 2063 μ g/ml expectorant heat, and 37 ℃ standing 24 hours; Supernatant discarded, with twice (remove and do not adhere to antibacterial) of 0.9% normal saline cleaning, then carry out matrix dyeing (extracellular matrix dyeing), PI dyeing (dead antibacterial is dyeed) and Syto9 dye (to or antibacterial dye), on laser confocal microscope, observe (PI, Syto9, the required excitation wavelength of matrix dyestuff are followed successively by 488nm, 559nm and 559nm), utilize imaris software to carry out image and data analysis, the results are shown in Figure 8.
Second group (penicillin sodium group): in little glass dish, add basic culture solution, then inoculate staphylococcus aureus, obtain 2mL initial OD
600nmvalue is 0.05 system, and 37 ℃ standing 24 hours; Reject supernatant, adds the basic culture solution containing 20 μ g/ml penicillin sodiums, and 37 ℃ standing 24 hours; Supernatant discarded, with twice (remove and do not adhere to antibacterial) of 0.9% normal saline cleaning, then carry out matrix dyeing (extracellular matrix dyeing), PI dyeing (dead antibacterial is dyeed) and Syto9 dye (to or antibacterial dye), on laser confocal microscope, observe (PI, Syto9, the required excitation wavelength of matrix dyestuff are followed successively by 488nm, 559nm and 559nm), utilize imaris software to carry out image and data analysis, the results are shown in Figure 8.
The 3rd group (matched group): in little glass dish, add basic culture solution, then inoculate staphylococcus aureus, obtain 2mL initial OD
600nmvalue is 0.05 system, and 37 ℃ standing 24 hours; Reject supernatant, adds basic culture solution, and 37 ℃ standing 24 hours; Supernatant discarded, with twice (remove and do not adhere to antibacterial) of 0.9% normal saline cleaning, then carry out matrix dyeing (extracellular matrix dyeing), PI dyeing (dead antibacterial is dyeed) and Syto9 dye (to or antibacterial dye), on laser confocal microscope, observe (PI, Syto9, the required excitation wavelength of matrix dyestuff are followed successively by 488nm, 559nm and 559nm), utilize imaris software to carry out image and data analysis, the results are shown in Figure 8.
By Fig. 8, can be observed: for ripe bacterial biof iotalm, expectorant heat can make clearly its viable bacteria greatly reduce, dead bacterium is increased greatly, and extracellular matrix reduces, and makes bacterial biof iotalm attenuation, smaller volume; For ripe bacterial biof iotalm, penicillin sodium is ineffective.This conclusion conforms to the testing result of microwell plate.Every group arranges 5 reprocessings.In each group, the result of 5 reprocessings is consistent.
By step 1 and step 2, can be drawn the following conclusions: penicillin sodium only has effect to the forming process of bacterial biof iotalm, ineffective to ripe bacterial biof iotalm, expectorant heat not only has effect to the forming process of bacterial biof iotalm clearly, and ripe bacterial biof iotalm is also had and suppressed or Degradation.
1, in BioFlux Plate outlet, add 200 μ L OD
600nmthe staphylococcus aureus bacterium liquid (bacterium liquid obtains with the resuspended staphylococcus aureus thalline of basic culture solution) of=0.6-0.8, is exported to Way in to power 5dyn/cm
2, 5min, standing 1h, discards the waste liquid of entrance and outlet.
2, at BioFlux Plate entrance, add 100 μ L OD
600nmthe staphylococcus aureus bacterium liquid (bacterium liquid obtains with the resuspended staphylococcus aureus thalline of basic culture solution) of=0.6-0.8, outlet adds 100 μ L OD
600nmthe staphylococcus aureus bacterium liquid (bacterium liquid obtains with the resuspended staphylococcus aureus thalline of basic culture solution) of=0.6-0.8, is exported to Way in to power 2dyn/cm
2, 2s, standing 2-4h, is covered with antibacterial in visible observation window under mirror, discards the waste liquid of entrance and outlet.
3, at BioFlux Plate entrance, add 1mL OD
600nmthe staphylococcus aureus bacterium liquid (bacterium liquid obtains with the resuspended staphylococcus aureus thalline of basic culture solution) of=0.6-0.8, outlet adds 100 μ L OD
600nmthe staphylococcus aureus bacterium liquid (bacterium liquid obtains with the resuspended staphylococcus aureus thalline of basic culture solution) of=0.6-0.8, entrance to Way out to power 0.15dyn/cm
2, cultivate 2-4d, form ripe biomembrane.
4, packet transaction
First group (expectorant heat clear group): at BioFlux Plate entrance, add 1mL to contain the clear basic culture solution of 2063 μ g/ml expectorant heat, entrance to Way out to power 0.15dyn/cm
2; Front half an hour, every 10min took pictures once, and after half an hour, every 30min takes pictures once, observed expectorant heat clearly on biomembranous impact;
Second group (penicillin sodium group): at BioFlux Plate entrance, add 1mL to contain the basic culture solution of 20 μ g/ml penicillin sodiums, entrance to Way out to power 0.15dyn/cm
2; Front half an hour, every 10min took pictures once, and after half an hour, every 30min takes pictures once, observed penicillin sodium to biomembranous impact.
For the environment of living in of biomembrane in analogue body, the present embodiment has adopted Bioflux shearing force model.Bioflux shearing force model is a kind of bacterium living beings membrane modle close to time of day, under accurate motive force effect, simulation antibacterial forms the dynamic process of bacterial biof iotalm in body, the bacterial biof iotalm forming is real bacterial biof iotalm, this model capable of dynamic, reacts the mechanism of medicine to bacterial biof iotalm intuitively.
Fig. 9 is shown in by photo after different time.Add after penicillin sodium, the state of bacterial biof iotalm and electron density and volume are all without obviously changing.Add after the clear 30min of expectorant heat the part between two spherical biofilms in pipeline to start to occur cavity, during 1h, biomembrane starts to peel off with pipeline, during 5h, biomembrane becomes loose, electron density and obviously diminishes, and during 10h, in pipeline, one, right side biomembrane dissipates, the biomembrane in left side cracks.The result of the present embodiment is consistent with microwell plate result and confocal laser scanning microscope result, has all illustrated that expectorant heat has destruction to ripe bacterial biof iotalm clearly, and penicillin sodium to ripe bacterial biof iotalm without effect.Every group arranges 5 reprocessings.In each group, the result of 5 reprocessings is consistent.
Claims (10)
1. expectorant heat is clear in the application of preparing in the biomembranous product of bacterium for degrading.
2. the expectorant heat application in the product of preparation destruction bacterial biof iotalm clearly.
3. the clear application in the product of preparing anti-bacteria biofilm formation of expectorant heat.
4. as the application as described in arbitrary in claims 1 to 3, it is characterized in that: described antibacterial is staphylococcus aureus.
5. as the application as described in arbitrary in claim 1 to 5, it is characterized in that: the clear dried frozen aquatic products for tanreqin injection or tanreqin injection of described expectorant heat.
6. the biomembranous product of bacterium for degrading, its active component is that expectorant heat is clear.
7. destroy a product for bacterial biof iotalm, its active component is that expectorant heat is clear.
8. a product for anti-bacteria biofilm formation, its active component is that expectorant heat is clear.
9. as the product as described in arbitrary in claim 6 to 8, it is characterized in that: described antibacterial is staphylococcus aureus.
10. as the product as described in arbitrary in claim 6 to 9, it is characterized in that: the clear dried frozen aquatic products for tanreqin injection or tanreqin injection of described expectorant heat.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201410005060.2A CN103720768A (en) | 2014-01-06 | 2014-01-06 | New use of tanreqing in preparation of product for destroying bacterial biofilm |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109925503A (en) * | 2017-12-19 | 2019-06-25 | 上海凯宝药业股份有限公司 | A kind of traditional Chinese medicine reversing drug resistance of Staphylococcus aureus |
| CN113546072A (en) * | 2021-07-12 | 2021-10-26 | 广西中医药大学 | Baicalein nanometer preparation and its preparing method and use |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101015600A (en) * | 2006-11-30 | 2007-08-15 | 浙江大学 | Desiccation process for tanreqin injection intermediates |
| KR20080002224A (en) * | 2006-06-30 | 2008-01-04 | (주)엠포엠 | Method of manufacturing shampoo having antibiotic activity against staphylococcus genus and shampoo composition therefrom |
-
2014
- 2014-01-06 CN CN201410005060.2A patent/CN103720768A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20080002224A (en) * | 2006-06-30 | 2008-01-04 | (주)엠포엠 | Method of manufacturing shampoo having antibiotic activity against staphylococcus genus and shampoo composition therefrom |
| CN101015600A (en) * | 2006-11-30 | 2007-08-15 | 浙江大学 | Desiccation process for tanreqin injection intermediates |
Non-Patent Citations (2)
| Title |
|---|
| 杨清林等: "痰热清注射液固形物中焦谷氨酸组分的RP-HPLC法测定", 《重庆师范大学学报(自然科学版)》, vol. 29, no. 5, 30 September 2012 (2012-09-30) * |
| 王毅: "痰热清注射液对细菌生物膜影响研究显示中药抗菌有优势", 《中国中医药报》, 3 January 2014 (2014-01-03) * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109925503A (en) * | 2017-12-19 | 2019-06-25 | 上海凯宝药业股份有限公司 | A kind of traditional Chinese medicine reversing drug resistance of Staphylococcus aureus |
| CN113546072A (en) * | 2021-07-12 | 2021-10-26 | 广西中医药大学 | Baicalein nanometer preparation and its preparing method and use |
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