CN101843631B - Application of baicalin in resisting and preventing bacterial infection - Google Patents
Application of baicalin in resisting and preventing bacterial infection Download PDFInfo
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- CN101843631B CN101843631B CN2010102020232A CN201010202023A CN101843631B CN 101843631 B CN101843631 B CN 101843631B CN 2010102020232 A CN2010102020232 A CN 2010102020232A CN 201010202023 A CN201010202023 A CN 201010202023A CN 101843631 B CN101843631 B CN 101843631B
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Abstract
The invention relates to application of baicalin in resisting and preventing bacterial infection and provides application of baicalin which does not bring survival pressure to bacteria and has little probability of generating resistance to drugs in resisting and preventing bacterial infection.
Description
Technical field
The invention belongs to biological technical field, relate in particular to the application of baicalin in bacterial-infection resisting and prevention of bacterial infection.
Background technology
The generation of resistance has limited the ability that people treat bacterial infection disease, and human health caused serious threat.Along with the continuous appearance of multiple Resistant strain, make traditional antibiotic face very big challenge to the treatment of bacterial infection disease.The reason that causes multi-drug resistant pathogen emerge in multitude on the one hand since in the last few years people antibiotic blindness abuse, on the other hand owing to the mechanism of action of traditional antibiotic itself.Because traditional antibiotic is to be action target spot with growth that suppresses antibacterial or existence, this survives for antibacterial itself and has brought very big pressure, thereby cause the generation of bacterial drug resistance.Penicillin for example, chloromycetin etc.Drug-fast constantly the spreading of pathogen makes a lot of traditional antibiotic in the treatment of clinical disease, lose effect, and people are very urgent to the demand of new antibiotic.Therefore seeking new antibiotic is one of very critical problem of current biology and medical circle.
The pathogenicity of antibacterial is its infection host and the ability that causes disease; And the pathogenicity of antibacterial depends on virulence factor that antibacterial produces and damage host's thereof mechanism of action; Pathogen is destroyed the normal physiological function of host cell through release endotoxin albumen, even causes the death of host cell.The pathogenicity of antibacterial is come the development of new antibiotic as target spot, and it is pathogenic but the growth of antibacterial is not exerted an influence the generation that causes bacterial drug resistance that therefore can be less to suppress antibacterial through this approach.In addition; All there is the quorum sensing system in the discovered in recent years various bacteria; (quorum sensing is to be semiochemicals with the diffusibility micromolecule QS), regulates and control that genes of individuals is expressed and group behavior a kind of important and comprehensive regulatory mechanism according to flora density in the quorum sensing system.The QS system is present in the middle of a lot of antibacterials, and modification scope has been contained the phenotype of a series of characteristic of bacteria property of antibacterial virulence factor, cytotoxin, biofilm formation or the like.So the quorum sensing system related gene of antibacterial and receive the expression of the downstream virulence factor of its regulation and control that the pathogenic course of antibacterial is played key effect has very important relation with the antibacterial pathogenicity.Therefore suppress the expression of the antibacterial QS system and the xicity related factor thereof, can have bigger potentiality to be exploited not damaging the action target spot that enlarges the Drug therapy bacterial infection disease under the human body self normal flora situation.
Pseudomonas aeruginosa (PAO) is a kind of very important opportunist, is considered to hospital infection property disease three big one of bacterium that cause death.For example suffer from the crowd of chronic disease in the low situation of body immunity, the patient of organ transplantation or patient of large-area burns or the like, it can cause various acute or chronic infection even cause death.There is very high inherent drug resistance in Pseudomonas aeruginosa itself; And making PAO produce serious drug resistance under the secular antibiotic therapy environment to many antibiotic, this pathogen that Pseudomonas aeruginosa is not only become be difficult to treat also makes it become the pathogenic and drug-fast object of numerous research antibacterials.
Chinese medicine has the medication history in thousands of years in China, and the ability of its disease preventing and treating is known by people, and Chinese medicine medical material kind is abundant, but wherein contains multiple Chinese medicine ingredients of not probed into and cure the disease mechanism.Modern Chinese medicine discovers that the chemical constituent of plurality of Chinese can be used as the natural antibacterial medicine and reaches antibiotic effect of curing the disease, and comprise the Flavonoid substances that plurality of Chinese contains, but its mechanism of action is unknown by the people.
Summary of the invention
In order to solve the above-mentioned technical problem that exists in the background technology, the invention provides the little application of baicalin in bacterial-infection resisting and prevention of bacterial infection of probability that baicalin brings survival pressure, develops immunity to drugs can not for antibacterial itself.
Technical solution of the present invention is: the application of baicalin in bacterial-infection resisting and prevention of bacterial infection.
Baicalin and its derivant and the application of trim in bacterial-infection resisting and prevention of bacterial infection that is synthesized as lead compound thereof.
The application of baicalin in bacterial-infection resisting medicine and prevention of bacterial infection medicine.
The application of baicalin in the prevention of bacterial with the medication combined medication of raising immunity catches medicine.
The application of baicalin in the acquired bacterial infective diseases medicine of prevention.
The application of baicalin in anti-gram-negative bacterial infections and prevention gram-negative bacterial infections medicine.
The application of baicalin in the medicine of medicine that resists gram-negative bacterial infections and the gram-negative bacterial infections that prevention has the QS system with QS system.
The application of baicalin in resisting pseudomonas aeruginosa infection medicine and prevention charrin disease medicine.
The application of baicalin in the medicine of the chronic pneumonia infection disease of treatment.
Baicalin is in the application of the medicine of eliminating biofilm.
Baicalin with the application of the bacterial-infection resisting medicine of antibiotic drug combination.
Advantage of the present invention is:
The present invention is from the pathogenic screening substances of bacterial resistance system, seeks new antibiotic and acts on the target site of antibacterial.Quorum sensing mortifier sieve nest system with making up has found that as the screening object flavones ingredient baicalin can suppress the pathogenicity of antibacterial effectively with Chinese medicine, and the Flavonoid substances baicalin is one of main chemical compositions of labiate Radix Scutellariae.And the present invention has finally found the molecular mechanisms of action of natural antibacterial medicine flavone; Be the flavones ingredient baicalin through to the bacterial community induction system with and the inhibition expressed of the virulence factor of level regulation and control weaken the pathogenic of antibacterial; It can suppress the pathogenicity of pathogen under the prerequisite of killing bacteria not itself; Bring survival pressure can for antibacterial itself,, can be used as novel anti antibacterial class medicine so the probability that develops immunity to drugs is less.
Description of drawings
Fig. 1 is the chemical molecular formula of baicalin;
Fig. 2 makes solid testing result sketch map for baicalin to the inhibition of 7 major genes of Pseudomonas aeruginosa quorum sensing system and other virulent genes;
Fig. 3 is the inhibitory action ELIASA testing result sketch map of baicalin to 7 major genes of Pseudomonas aeruginosa quorum sensing system and other virulent genes;
Fig. 4 is that baicalin is to the biological film formed sketch map that influences of Pseudomonas aeruginosa;
Fig. 5 is the influence sketch map of baicalin to the Pseudomonas aeruginosa pyo;
Fig. 6 is the influence sketch map of baicalin to the Pseudomonas aeruginosa elastoser;
Fig. 7 for baicalin to the Pseudomonas aeruginosa motor capacity: clump moving (a) and spring up the sketch map that influences of (b);
Fig. 8 influences the cytotoxicity sketch map for baicalin;
Fig. 9 kills the cell ability sketch map for baicalin suppresses Pseudomonas aeruginosa.
The specific embodiment
The invention provides the application of baicalin in bacterial-infection resisting and prevention of bacterial infection; Especially baicalin and its derivant and the trim that is synthesized as the lead compound application in bacterial-infection resisting and prevention of bacterial infect; The application of baicalin in bacterial-infection resisting medicine and prevention of bacterial infection medicine; The application of baicalin in the prevention of bacterial with the medication combined medication of raising immunity catches medicine; The application of baicalin in the acquired bacterial infective diseases medicine of prevention; The application of baicalin in anti-gram-negative bacterial infections and prevention gram-negative bacterial infections medicine; The application of baicalin in the medicine of medicine that resists gram-negative bacterial infections and the gram-negative bacterial infections that prevention has the QS system with QS system, the application of baicalin in resisting pseudomonas aeruginosa infection medicine and prevention charrin disease medicine, the application of baicalin in the medicine of the chronic pneumonia infection disease of treatment; Baicalin is in the application of the medicine of eliminating biofilm, baicalin with the application of the bacterial-infection resisting medicine of antibiotic drug combination.
It is screening system for the Pseudomonas aeruginosa toxicity correlation factor of reporter starts word bank that utilization of the present invention contains luciferase (Luciferase) gene (lux-CDABE); Utilize solid double-layer medium therapy and enzyme mark detector that the alcohol of the conventional Chinese medicine that is used for " heat-clearing and toxic substances removing " is traditionally mentioned that water extract screens, detect the variation of Pseudomonas aeruginosa toxicity related gene expression under the condition that the Chinese medicine medicine exists; And detected the variation of Pseudomonas aeruginosa and pathogenic relevant phenotype under drug effect simultaneously, like extracellular protease, the clump of pyo and antibacterial is moving and spring up or the like.Find that baikal skullcap root has the obvious suppression effect at the following of the condition that does not influence bacterial growth to Pseudomonas aeruginosa (PAO) is pathogenic; And then find the pathogenicity of Radix Scutellariae effective ingredient baicalin at the following obvious inhibition PAO of the condition that does not influence bacterial growth, and tradition research report baicalin has antibacterial, antiallergic action, blood pressure lowering and calmness, function of gallbladder promoting, protects the liver and effect such as spasmolytic.Because the existence to antibacterial does not produce selection pressure, thus generation can be less or that avoid causing bacterial drug resistance, and baicalin can be used as the medicinal application of novel anti mushroom.
Technical scheme provided by the present invention obtains through following scheme:
Embodiment 1:
The structure of the pathogenic screening substances of bacterial resistance system.Plasmid pMS402 is the carrier that has the reporter gene lux-CDABE that lacks promoter.The toxicity related gene comprises lasR, rhlI, rhlR, lasI, exoT, exsD, exoS, oprH, phzA1, phzA2, pilG, fliC, mig4, xcpR, aprA, rnr.The promoter region of gene is through pcr amplification, and through enzyme cutting clone before report plasmid pMS402 luciferase gene operon, the recombiant plasmid that builds up is called after pKD-lasR respectively, pKD-rhlI, pKD-rhlR; PKD-lasI, pKD-exoT, pKD-exsD, pKD-exoS, pKD-oprH; PKD-phzA 1, pKD-phzA2, pKD-pilG, pKD-fliC; PKD-migA, pKD-xcpR, pKD-aprA, pKD-rnr.With recombiant plasmid with the PacI enzyme action after big fragment contain the promoter sequence of lux reporter gene and toxicity related gene; Size is about about 8kb; Use PacI digested plasmid CTX6.1 simultaneously, this plasmid phage binding site capable of using carries out site-specific nature reorganization enzyme action.With purification good have promoter-reporter fusant lux-CDABE fragment cloning in the CTX6.1 carrier, obtain size and be the called after pKD-CTX-lasR respectively of the recombiant plasmid about 14kb, pKD-CTX-rhlI, pKD-CTX-rhlR; PKD-CTX-lasI, pKD-CTX-exoT, pKD-CTX-exsD, pKD-CTX-exoS; PKD-CTX-oprH, pKD-CTX-phzA1, pKD-CTX-phzA2; PKD-CTX-pilG, pKD-CTX-fliC, pKD-CTX-m igA; PKD-CTX-xcpR, pKD-CTX-aprA, pKD-CTX-rnr.This recombiant plasmid electricity is transformed in the 10B competent cell cultivates; To extract the great deal of high concentration plasmid; Change among the SM10, SM10 and the wild type PAO1 that will contain recombiant plasmid again carry out parents' strain hybridization again, and the fusant of promoter-reporter is integrated in chromosome.Reporter gene lux-CDABE does not have self promoter, expresses the regulation and control receive the QS related gene promoter, and when medicine then to be measured had inhibitory action to the promoter of PAO virulence factor, then lux gene expression also can weaken, i.e. the thalline luminescent decay; Otherwise, if testing sample has activation to promoter, the then luminous enhancing of thalline.Can know the situation that influences that medicine to be measured is expressed virulence factor through the apparent upward luminous variation of thalline.
Embodiment 2:
The solid plate screening:
The double-deck basic point filter paper method of cultivating of utilization is carried out the solid scalping: find that baicalin has the effect that suppresses Pseudomonas aeruginosa quorum sensing system and other a plurality of virulence factors expression, its chemical molecular formula is as shown in Figure 1.Under the same conditions, these samples do not have influence basically to bacterial growth, enumerate baicalin to QS related gene lasR, rhlI, rhlR and lasI; Three type excretory system gene exoT; ExoS, the dull and stereotyped figure that detects of the inhibition that azophenlyene class synthetic gene phzA1 expresses sees Fig. 2.
Embodiment 3:
Enzyme mark detector method is carried out liquid and is sieved again: analyze the scalping result, utilize enzyme mark detector in 96 orifice plates, various Chinese medicinal ingredients to be carried out multiple sieve, find baicalin, have the Pseudomonas aeruginosa quorum sensing system of inhibition and the luminous effect of other a plurality of virulence factors.
Referring to Fig. 3 A (a), suppress effect and when the concentration that contains baicalin is 250 μ g/mL, the inhibitory action that the self-induction thing synthase gene rhlI of quorum sensing Rhl system expresses is reached more than 3 times; Referring to Fig. 3 B (a) inhibitory action of lasR gene expression is reached 2 times; Referring to Fig. 3 C (a) and Fig. 3 D (a), to rhlR, the inhibitory action of lasI gene expression is less than 2 times; Referring to Fig. 3 E (a), the inhibitory action of phzA1 gene expression is reached more than 8 times; Referring to Fig. 3 F (a), the inhibitory action of exoS gene expression is reached more than 5 times; Referring to Fig. 3 G (a), the inhibitory action of exoT gene expression is reached 2 times; Discover that baicalin all has the obvious suppression effect to four genes of bacterial community induction system and other virulent genes; In addition all the baicalin experimental port is luminous change in; Compare all growth curve charts shows with contrast; Baicalin is to the not influence of growth of antibacterial, respectively referring to Fig. 3 A (b), Fig. 3 B (b), Fig. 3 C (b), Fig. 3 D (b), Fig. 3 E (b), Fig. 3 F (b) and Fig. 3 G (b).
Embodiment 4:
Influence detects baicalin to Pseudomonas aeruginosa virulence factor phenotype:
Pseudomonas aeruginosa comes infection host to cause the host to be caused a disease through producing a large amount of virulence factors; And the QS system of Pseudomonas aeruginosa almost regulates the expression of all virulence factors; Comprise protease (elastoser, alkaline protease), pyo, hemolysin; Exoenzyme S and extracellular toxin A or the like.
The virulence factor of testing applied screening system comprises phzA1, phzA2, lasI, lasR, rhlI, rhlR, fliC, pilG, oprH, aprA, exoS, exoT, exsD, rnr, xcpR, migA, and wherein lasR, rhlR are that upper reaches controlling gene and lasI, the rhlI of Pseudomonas aeruginosa quorum sensing system (QS) are the synthetic catalyzing enzyme gene of signaling molecule.It all is the control that receives the numerator mediated intercellular signal system of quorum sensing system signal that the outer virulence factor of necessary multiple born of the same parents is organized in charrin disease host invasion; These regulator control systems make Pseudomonas aeruginosa can produce the outer virulence factor of born of the same parents with the mode of coordinating, cell density relies on, thereby defeat host's system of defense.Comprise three type excretory system (T3SS) related gene exoS, exoT, exsD in addition; The fliC relevant, pilG with bacterial motility; The motor capacity of antibacterial is relevant with antibacterial pathogenecity in vivo, and is also relevant with biomembranous formation; Azophenlyene synthetic gene phzA1, phzA2; And with epicyte protein oprH, channel protein xcpR; Relevant enzymes glycosyl transferase migA, ribonuclease rnr, alkaline protease aprA, the expression of these virulence factors is played important effect to the Pseudomonas aeruginosa pathogenic course.
(1) Pseudomonas aeruginosa exists with the form of biofilm in host mostly; So-called biofilm refers to antibacterial when being adsorbed in material surface; Be membranaceous many bacterium aggregation (ommunity) structure by what the outer poly material of the secreted born of the same parents of antibacterial and antibacterial was formed; For in its growth or process, adapt to extraneous the killing and wounding of living environment opposing like antibiotic substance at host cells infected.Many discovering forms biofilm form PA thalline with respect to the somatic cells that exists with free form, can improve the Drug resistance of Pseudomonas aeruginosa significantly, can make thalline resist the immune system of extraneous drug effect and escape host body.Concrete scheme is: choose the PA monoclonal to 5mL LB fluid medium; The bacteria suspension and the 3 μ L baicalin DMSO solution (96 orifice plate) in the DMEM culture medium that add 5 μ L dilution; Final volume is 150 μ L, and the baicalin ultimate density is 125 μ g/mL, and parallel laboratory test group (dosing) and parallel matched group (adding DMSO) are set; 37 ℃, concussion 200rpm, 3h; Place 30 ℃ again and leave standstill and wash the liquid hole in off after cultivating 48h, add 150 μ L 0.5%CV (crystal violet) solution to each hole then, cultivate 5min under the room temperature.After washing crystal violet off; Carefully fall unconjugated crystal violet with distilled water flushing; Oven dry adds the alcoholic solution of 150 μ L 95% then in the hole in baking oven, surveys its light absorption value in the 570nm wavelength then, referring to Fig. 4; In the presence of the medicine under collating condition, the formed biofilm biomass of PA obviously reduces.
(2) pyo is the excretory a kind of important destruction cell of Pseudomonas aeruginosa, the azophenlyene class extracellular toxin of tissue.Pyo is measured scheme: choose the PA monoclonal to 5mL LB fluid medium, 37 ℃ of experimental group (dosing) and matched groups (adding DMSO) are set, concussion 200rpm cultivates 14h.Incubated overnight bacterium liquid is centrifugal, and centrifugal 2 minutes of 8000rpm gets supernatant and changes in another centrifuge tube, adds the extracting of 3mL chloroform in every 5mL supernatant, centrifugal 8 minutes of 4500rpm.Take off layer chloroform layer and change in the new centrifuge tube, and add the mixed in hydrochloric acid of 1mL 0.2N.Abundant mixing, the centrifugal and inorganic phase in collection upper strata is surveyed its light absorption value in the 520nm wavelength.Referring to Fig. 5, obviously reduce in the pyo amount that PA produced under the collating condition under the baicalin effect.
(3) the excretory elastoser of Pseudomonas aeruginosa has the ability of multiple proteins such as destroying host's collagen protein, elastin laminin, also is that PA causes one of morbific key factor of body.Elastin laminin enzymatic determination scheme: picking PA monoclonal is to 5mL LB fluid medium; Experimental group (dosing) and 37 ℃ of matched groups (adding DMSO), concussion 200rpm are set, cultivate OD=2.0; The bacteria suspension of cultivating is centrifugal; Supernatant with 0.2 μ m membrane filtration, is mixed supernatant fluid filtrate and phosphate buffer (0.1M 6.3PH) and adds 2mg/mLelastin Congo red (Sigma) at 2: 1, the zeroing group is set promptly has only culture medium and Congo red albumen.37 ℃ in mixture, concussion 200rpm, behind the cultivation 7d, the centrifuging and taking supernatant is surveyed OD under 495nm.Referring to Fig. 6, in the presence of baicalin, the elastin laminin amount that PA produced obviously reduces than collating condition.
(4) flagellum of mobility of antibacterial and antibacterial is closely related, in the antibacterial pathogenic course, has the important pathological meaning, and the mobility of antibacterial is also relevant with biomembranous formation in addition.Bacterioflora moves and spring up the mensuration scheme of ability: picking PA monoclonal 37 ℃, shakes 200rpm, overnight incubation to 5mL LB fluid medium.The preparation antibacterial springs up flat board and clump moving dull and stereotyped (placement is used after spending the night).Treat that flat board is divided into equal zone, the filter paper of a diameter 6mm is placed in each district, with Chinese medicine medicine lyophilization powder in the water-soluble or methanol; The pure article of Chinese medicine are dissolved among the DMSO and mix with bacteria suspension, click and enter the sample solution of a series of diluted concentrations such as 1,1/2,1/4,1/8 of 5 μ L respectively; With 5 μ L ddH2O/ methanol/DMSO as negative control; Referring to Fig. 7, under collating condition, the motor capacity of PA obviously weakens in the presence of medicine.
Embodiment 6:
Mtt assay detects the baicalin cytotoxicity and suppresses Pseudomonas aeruginosa and kill cell ability.
Collect the logarithmic (log) phase cell, the adjustment concentration of cell suspension, every hole adds 196 μ L (detection of baicalin cytotoxicity) or 191 μ L (baicalin suppresses Pseudomonas aeruginosa and kills the cell ability detection), and it is 10 that bed board makes cell density to be measured
3-10
4/ hole; 5%CO
2, 37 ℃ hatch be paved with the hole to cell at the bottom of (96 orifice plate), add 4 μ L gradient concentration medicines behind the 24h or contrast adds equivalent 4 μ L DMSO, baicalin suppresses Pseudomonas aeruginosa and kills cell ability and detect and add 5 μ L bacteria suspensions or contrast adds 5 μ LDMEM culture medium; 5%CO
2, cultivate 16-48h for 37 ℃, inverted microscope is observed down; Every hole adds 20 μ LMTT solution, and (5mg/mL 0.5%MTT) continues to cultivate 4h; Stop cultivating, the careful suction removed culture fluid in the hole; Every hole adds 150 μ L DMSO, low speed concussion 10min, and crystal fully dissolves the OD=490nm place surveys light absorption value, and referring to Fig. 8, cytotoxicity experiment proof baicalin does not influence hepatocellular normal growth, no cytotoxicity at concentration 250 μ g/mL; Referring to Fig. 9, baicalin suppresses PA and kills cell ability detection discovery, kills cell ability than collating condition PA in the presence of baicalin and obviously is suppressed.
Claims (1)
1. baicalin is as the application of unique active component in preparation resisting pseudomonas aeruginosa infection medicine and prevention charrin disease medicine, and the concentration of baicalin is 250 μ g/mL in this medicine.
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| CN105287622B (en) * | 2015-10-23 | 2019-05-17 | 陕西省微生物研究所 | NO accumulation reduces method, target spot and the application of pseudomonas aeruginosa invasiveness |
| CN105796584B (en) * | 2016-04-18 | 2019-01-25 | 安徽延寿堂药业有限公司 | A kind of medicinal liquid injection of lincomycin hydrochloride composition and its preparation method of formula and pharmaceutical preparation |
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