CN105998153A - Application of pithecellobium clypearia extract to preparation of drug for resisting generation of extended spectrum beta-lactamase escherichia coli - Google Patents
Application of pithecellobium clypearia extract to preparation of drug for resisting generation of extended spectrum beta-lactamase escherichia coli Download PDFInfo
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Abstract
The invention discloses application of pithecellobium clypearia extract to preparation of a drug for resisting generation of extended spectrum beta-lactamase (ESBL) escherichia coli, further discloses application of pithecellobium clypearia extract to preparation of a sensibilization drug for resisting generation of extended spectrum beta-lactamase escherichia coli with antibiotics, and particularly relates to application to a drug for resisting generation of extended spectrum beta-lactamase escherichia coli as amikacin and sulfamethoxazole. It is proved through tests that the pithecellobium clypearia extract and amikacin or sulfamethoxazole take the synergistic effect when combined. The pithecellobium clypearia extract is combined when smaller than or equal to 1/2MIC, and the usage is lower than that of amikacin used singly by 75% and is lower than that of sulfamethoxazole used singly by 99.3%. The drug can be used as a natural antibacterial drug or antibiotic sensitizer and is applied to treat diseases caused by generation of ESBL escherichia coli. A new path and a substitute drug are provided for solving the drug resistance problem of the antibiotics.
Description
Technical field
The invention belongs to field of traditional Chinese medicine pharmacy, relate to Ramulus Et Folium Pithecellobii Lucidi (Pithecellobium clypearia) and carry
Take the new application of thing.
Background technology
21 century is the epoch of multi-drug resistant bacteria, after antibiotic clinical practice 60 years, and increasing institute
Interior infection and multi-drug resistant bacteria infect a great problem becoming the most clinical antibacterial therapy, Jerons in 1961
Reporting the 1st example clinic methicillin-resistant staphylococcus aureus (MRSA) infected patient, current MRSA feels
Dye the most gradually extends over the entire globe；The distribution of main fastbacteria in Chinese bacterial drug resistance monitoring clinic in 2011,
Escherichia coli, Klebsiella Pneumoniae ESBLs-producing bacteria (ESBL) strain average out to 50.7% respectively,
38.5%, transition and the present situation of its drug resistance receive much concern.It addition, according to China's Ministry of Public Health Surveillance on antibiotic resistance cooperation
Organize 2010 annual reports, Acinetobacter bauamnnii and Pseudomonas aeruginosa in the pathogen that hospital ICU separates
Ranking first, Acinetobacter bauamnnii is up to 60.4% He respectively to the resistant rate of imipenum and meropenem
61.4%.In sum, at MRSA, each bacterioid of ESBL, multidrug resistant Acinetobacter bauamnnii are produced
Rise year by year with the clinical occupation rate of Pseudomonas aeruginosa, under the pressure that resistant rate is constantly climbed to a higher point, antibiotic
Treatment is faced with huge challenge.For avoiding the further deterioration of bacterial resistance phenomenon, experts and scholars try hard to
Find that new bacteria growing inhibiting and treatment antibacterial cause the new method of disease.Existing research report proves
Chinese tradition Chinese medicine such as Rhizoma Coptidis, Radix Scutellariae and Fructus Forsythiae etc. have certain inhibition to different drug-resistant bacterias, enter
What one step Study of Traditional Chinese Medicine suppression fastbacteria grew focuses on finding that new bacteriostasis is higher, drug resistance is antibacterial
Compose wider array of Chinese medicine.
Ramulus Et Folium Pithecellobii Lucidi (Pithecellobium clypearia Benth), formal name used at school bib tree, is Mimosaceae
The dry sprout of Ramulus Et Folium Pithecellobii Lucidi platymiscium Ramulus Et Folium Pithecellobii Lucidi and leaf, its nature and flavor bitterness is cold, effect heat-clearing and toxic substances removing, removing dampness
Sore, is to treat the southern medical material that multiple pyretic toxicity disease is unique.
The open Ramulus Et Folium Pithecellobii Lucidi of document and its extract is had to have antivirus action at present.Chinese patent
CN103385912A discloses the extract of Ramulus Et Folium Pithecellobii Lucidi and has anti-MRSA effect and antibiotic sensitization,
But ESBLs-producing bacteria escherichia coli (are called for short and " produce by this patent this Ramulus Et Folium Pithecellobii Lucidi extract not mentioned
ESBL escherichia coli ") antibacterial action and with antibiotic associated with sensitization.
Summary of the invention
Present invention aim at developing Ramulus Et Folium Pithecellobii Lucidi application in preparing drug-resistance bacteria medicine.
The invention discloses Ramulus Et Folium Pithecellobii Lucidi extract and prepare anti-ESBLs-producing bacteria escherichia coli medicine
In application.
And disclose Ramulus Et Folium Pithecellobii Lucidi extract further and preparing antibiotic anti-ESBLs-producing bacteria large intestine
Application in bacillus hypersitization medicine.Especially as the anti-product of amikacin and bactrim surpass wide spectrum β-
Application in lactamase escherichia coli hypersitization medicine.Present invention can apply to human medicine or animal-use drug
Thing.
Ramulus Et Folium Pithecellobii Lucidi extract of the present invention refers to Ramulus Et Folium Pithecellobii Lucidi water extract or Ramulus Et Folium Pithecellobii Lucidi ethanol extraction.
This extract can be prepared by the following method: Ramulus Et Folium Pithecellobii Lucidi coarse powder water or ethanol water extract, gained
Extracting solution is extracted with ethyl acetate the extract of gained again and is target product.
The beneficial effects of the present invention is: present invention firstly discloses Ramulus Et Folium Pithecellobii Lucidi extract to producing ESBL large intestine
The antibacterial action of bacillus and the sensitization to such antibiotic.Tests prove that, Ramulus Et Folium Pithecellobii Lucidi extract with
Amikacin or bactrim are combined all in synergism.It is less than or equal at Ramulus Et Folium Pithecellobii Lucidi extract of the present invention
Being combined during 1/2MIC, consumption more alone than amikacin reduces by 75%；Consumption more alone than bactrim reduces
99.3%.Can be applied to by producing ESBL escherichia coli as natural antibacterials or antibiotic sensitizer
The disease treatment caused.Provide new approach for solving the drug resistance problems of such antibiotic and substitute medicine
Thing.
Detailed description of the invention
The present invention by following embodiment to Ramulus Et Folium Pithecellobii Lucidi water or the anti-product ESBL large intestine bar of ethanol extraction
The pharmacological action of bacterium is screened.
Colibacillary minimum antibacterial dense to producing ESBL with micro-broth dilution method Ramulus Et Folium Pithecellobii Lucidi extract
Degree (MIC) and minimum bactericidal concentration (MBC).
Bacterial strain: produce ESBL escherichia coli (ECO, numbering E1-E20) 20 strains；Escherichia coli Quality-control strains
(ECO, ATCC25922) is by No.1 Hospital Affiliated to Zhongshan Univ.'s laboratory medicine portion clinical microbiology laboratory technique
Room provides, and through No.1 Hospital Affiliated to Zhongshan Univ.'s clinical microbiology laboratory technique room, detection confirms its drug resistance.
MH broth bouillon: MH meat soup dry powder (Britain OXOID LTD.) 2.1g, is settled to 100ml,
NAOH regulates pH to 7.0, autoclaving, puts 4 DEG C of refrigerators standby.
Method: the trace meat soup recommended with reference to US National clinical trial standardization committee (NCCLS) is dilute
Interpretation of the law operates.
The present invention by following method to Ramulus Et Folium Pithecellobii Lucidi extract to amikacin, bactrim anti-product ESBL
Colibacillary sensitization is investigated.
Bacterial strain: produce ESBLs escherichia coli (ECO, numbering E1-E20) 20 strains；Escherichia coli Quality Control bacterium
Strain (ECO, ATCC25922) is by No.1 Hospital Affiliated to Zhongshan Univ.'s laboratory medicine portion Clinical microorganism inspection
Testing room to provide, through No.1 Hospital Affiliated to Zhongshan Univ.'s clinical microbiology laboratory technique room, detection confirms its drug resistance.
MH broth bouillon: MH meat soup dry powder (Britain OXOID LTD.) 2.1g, is settled to 100ml,
NAOH regulates pH to 7.0, autoclaving, puts 4 DEG C of refrigerators standby.
Method: the checkerboard method recommended with reference to US National clinical trial standardization committee (NCCLS)
Operation.
The preparation method of Ramulus Et Folium Pithecellobii Lucidi extract: Ramulus Et Folium Pithecellobii Lucidi (Pithecellobium clypearia Benth)
Thered is provided by Guangzhou Huacheng pharmaceutical plant.Take appropriate pithecellobium clypearia and break into coarse powder, with water or alcohol reflux 2
Secondary, each 2 hours, filter；Merging filtrate, is concentrated to give extractum (water or ethanol extraction).Take extractum
After water suspendible, it is extracted with ethyl acetate, extracts three times, combined ethyl acetate extract, be concentrated to give second
Acetoacetic ester extract.Described ethanol extraction can be prepared by 10-95% alcohol reflux.
Technical scheme is further illustrated below by specific embodiment.
Embodiment 1
The acetic acid ethyl ester extract anti-product ESBL colibacillary bacteriostasis and sterilization test of Ramulus Et Folium Pithecellobii Lucidi water extract
And respectively with amikacin, bactrim united enhanced sensitivity Effect tests.
1. experimental technique
1) mensuration of minimum inhibitory concentration (MIC):
The acetic acid ethyl ester extract of Ramulus Et Folium Pithecellobii Lucidi water extract, amikacin (AMK), bactrim (SXT)
Carrying out a series of doubling dilution, every hole 50 μ l respectively in MH broth bouillon, regulation inoculation bacterium is
1.0×106CFU/ml, every hole 50 μ l bacterium solution.35 DEG C of cultivations；24 hours, without precipitating the minimum of appearance
The concentration of antibacterials is its minimum inhibitory concentration (MIC).
2) mensuration of minimum bactericidal concentration (MBC):
Use flat board coating counting method, from 1) 50ul bacteria suspension is drawn in the hole of item asepsis growth put down to blood
On plate, even spread, cultivate 24 hours for 35 DEG C, colony counting, make initial experiment viable count reduce
The concentration of 99.9% or above required minimum antibacterials is its minimum bactericidal concentration (MBC).
By measuring minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of medicine, and to data
Carry out statistics and draw MIC50, MIC90, MBC50, MBC90, evaluate medicine anti-product ESBL large intestine bar
The effect of bacterium.
3) checkerboard method:
Checkerboard method is carried out in 96 hole aseptic culture plates, the ethyl acetate of Ramulus Et Folium Pithecellobii Lucidi water extract is extracted
Take thing and amikacin (AMK), bactrim (SXT) respectively in MH broth bouillon multiple proportions dilute
Being interpreted into series concentration, combine respectively with two medicines each 1/4MIC to 4MIC, every hole A medicine B medicine is each
Adding 25 μ L, the concentration adjusting bacteria suspension is 1.0 × 106CFU/ml, every hole inoculates 50 μ L bacterium solution, 35 DEG C
To producing the colibacillary minimum inhibitory concentration of ESBLs (MIC) after observing the associating of A medicine B medicine after hatching 24h.
The calculating of FIC index:Antibacterial by calculating section
Index (FIC) evaluates the acetic acid ethyl ester extract of Ramulus Et Folium Pithecellobii Lucidi water extract and the phase that Antibiotic combination is antibacterial
Interaction, FIC≤0.5 is synergism, and 0.5 FIC≤1 is summation action, and 1 FIC≤2 are nothing
Pass acts on, and FIC 2 is antagonism；And the acetic acid of Ramulus Et Folium Pithecellobii Lucidi water extract is found out according to asepsis growth hole
Ethyl ester extract and the best concentration ratio of antibiotic, the final ethyl acetate evaluating Ramulus Et Folium Pithecellobii Lucidi water extract
Extract strengthens the effect of antibiotic effect.
2. experimental result
The acetic acid ethyl ester extract of Ramulus Et Folium Pithecellobii Lucidi water extract and two kinds of antibiotic (new promises of amikacin, compound recipe
Bright) the product colibacillary extracorporeal bacteria inhibitor test of ESBL be the results are shown in Table 1.
The acetic acid ethyl ester extract of Ramulus Et Folium Pithecellobii Lucidi water extract is to producing the test of ESBLs colibacillary extracorporeal disinfecting
The results are shown in Table 2.
Through statistical analysis, the acetic acid ethyl ester extract of Ramulus Et Folium Pithecellobii Lucidi water extract and two kinds of antibiotic to product
MIC50, MIC90 of the colibacillary In Vitro Bacteriostasis of ESBL and sterilization are shown in Table 3.
Through statistical analysis, Ramulus Et Folium Pithecellobii Lucidi extract MBC50, MBC90 to producing the colibacillary sterilization of ESBL
It is shown in Table 4.
The FIC of the Combination susceptibility testing of the acetic acid ethyl ester extract of Ramulus Et Folium Pithecellobii Lucidi water extract and two kinds of antibiotic
The distribution statistics of value and FIC value the results are shown in Table 5, table 6.
The acetic acid ethyl ester extract of Ramulus Et Folium Pithecellobii Lucidi water extract to after the sensitization of two kinds of antibiotic and combination its
MIC50, MIC90 are shown in Table 7-9.
Table 1
Table 2
Table 3
Table 4
Table 5
Table 6
Table 7
Table 8
Table 9
Test result indicate that the acetic acid ethyl ester extract of Ramulus Et Folium Pithecellobii Lucidi water extract is alone to producing ESBL large intestine
The MIC50 of bacillus is 1600 μ g/ml, and MIC90 is 1600 μ g/ml, and MBC50 is 3200 μ g/ml,
MBC90 is 3200 μ g/ml；
20 strains are produced ESBL escherichia coli, the acetic acid ethyl ester extract of Ramulus Et Folium Pithecellobii Lucidi water extract and Ah rice
FIC associated with card star all≤2 shows that two medicines, without antagonism, wherein have 50%FIC value≤0.5 in association
Same-action；The acetic acid ethyl ester extract of Ramulus Et Folium Pithecellobii Lucidi water extract makes A meter Ka when less than or equal to 1/2MIC
Star MIC50 is down to 4 μ g/ml from 16 alone μ g/ml, reduces 75%；MIC90 is from alone
64 μ g/ml are down to 16 μ g/ml, reduce by 75%；
20 strains are produced ESBL with bactrim combination by the acetic acid ethyl ester extract of Ramulus Et Folium Pithecellobii Lucidi water extract
Escherichia coli FIC all≤2 show two medicine two medicines without antagonism, wherein 25%FIC value≤0.5 is in association
Same-action.The acetic acid ethyl ester extract of Ramulus Et Folium Pithecellobii Lucidi water extract makes compound recipe when less than or equal to alone MIC
Sulfamethoxazole MIC50 and MIC90 is all down to 19/1 μ g/ml from 2432/128 alone μ g/ml, fall
Low by 99.3%.
Embodiment 2
The acetic acid ethyl ester extract anti-product colibacillary bacteriostasis and sterilization of ESBL of Ramulus Et Folium Pithecellobii Lucidi 10% ethanol extraction
Test and respectively with amikacin, bactrim united enhanced sensitivity Effect tests.
1. experimental technique
1) mensuration of minimum inhibitory concentration (MIC):
The acetic acid ethyl ester extract of Ramulus Et Folium Pithecellobii Lucidi 10% ethanol extraction, amikacin (AMK), the new promise of compound recipe
Bright (SXT) carries out a series of doubling dilution, every hole 50 μ l, regulation respectively in MH broth bouillon
Inoculation bacterium is 1.0 × 106CFU/ml, every hole 50 μ l bacterium solution.35 DEG C of cultivations；24 hours, without being settled out
The concentration of existing minimum antibacterials is its minimum inhibitory concentration (MIC).
2) mensuration of minimum bactericidal concentration (MBC):
Use flat board coating counting method, from 1) 50ul bacteria suspension is drawn in the hole of item asepsis growth put down to blood
On plate, even spread, cultivate 24 hours for 35 DEG C, colony counting, make initial experiment viable count reduce
The concentration of 99.9% or above required minimum antibacterials is its minimum bactericidal concentration (MBC).
By measuring minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of medicine, and to data
Carry out statistics and draw MIC50, MIC90, MBC50, MBC90, evaluate medicine anti-product ESBL large intestine bar
The effect of bacterium.
3) checkerboard method:
Checkerboard method is carried out, by the acetic acid of Ramulus Et Folium Pithecellobii Lucidi 10% ethanol extraction in 96 hole aseptic culture plates
Ethyl ester extract and amikacin (AMK), bactrim (SXT) are respectively in MH broth bouillon
Doubling dilution becomes series concentration, combines respectively with two medicines each 1/4MIC to 4MIC, every hole A medicine
B medicine respectively adds 25 μ L, and the concentration adjusting bacteria suspension is 1.0 × 106CFU/ml, every hole inoculates 50 μ L bacterium solution,
35 DEG C hatch observe the associating of A medicine B medicine after 24h after to producing the colibacillary minimum inhibitory concentration of ESBLs
(MIC)。
The calculating of FIC index:Antibacterial by calculating section
Index (FIC) is evaluated the acetic acid ethyl ester extract of Ramulus Et Folium Pithecellobii Lucidi 10% ethanol extraction and is resisted with Antibiotic combination
The interaction of bacterium, FIC≤0.5 is synergism, and 0.5 FIC≤1 is summation action, 1 FIC≤2
For unrelated effect, FIC 2 is antagonism；And find out Ramulus Et Folium Pithecellobii Lucidi 10% ethanol according to asepsis growth hole and carry
Take the acetic acid ethyl ester extract of thing and the best concentration ratio of antibiotic, final evaluation Ramulus Et Folium Pithecellobii Lucidi 10% ethanol
The acetic acid ethyl ester extract of extract strengthens the effect of antibiotic effect.
2. experimental result
The acetic acid ethyl ester extract of Ramulus Et Folium Pithecellobii Lucidi 10% ethanol extraction and two kinds of antibiotic are (amikacin, multiple
Side's sulfamethoxazole) the product colibacillary extracorporeal bacteria inhibitor test of ESBL be the results are shown in Table 10.
To producing, ESBLs is colibacillary external to be killed the acetic acid ethyl ester extract of Ramulus Et Folium Pithecellobii Lucidi 10% ethanol extraction
Bacterium result of the test is shown in Table 11.
Through statistical analysis, the acetic acid ethyl ester extract of Ramulus Et Folium Pithecellobii Lucidi 10% ethanol extraction and two kinds of antibiotic
MIC50, the MIC90 producing the colibacillary In Vitro Bacteriostasis of ESBL and sterilization is shown in Table 12.
Through statistical analysis, Ramulus Et Folium Pithecellobii Lucidi extract MBC50, MBC90 to producing the colibacillary sterilization of ESBL
It is shown in Table 13.
The acetic acid ethyl ester extract of Ramulus Et Folium Pithecellobii Lucidi 10% ethanol extraction and the Combination susceptibility testing of two kinds of antibiotic
FIC value and the distribution statistics of FIC value the results are shown in Table 14, table 15.
The acetic acid ethyl ester extract of Ramulus Et Folium Pithecellobii Lucidi 10% ethanol extraction is to the sensitization of two kinds of antibiotic and connection
It is shown in Table 16-18 with rear its MIC50, MIC90.
Table 10
Table 11
Table 12
Table 13
Table 14
Table 15
Table 16
Table 17
Table 18
Test result indicate that the acetic acid ethyl ester extract of Ramulus Et Folium Pithecellobii Lucidi 10% ethanol extraction is alone to producing ESBL
Colibacillary MIC50 is 1600 μ g/ml, and MIC90 is 1600 μ g/ml, and MBC50 is
3200 μ g/ml, MBC90 are 3200 μ g/ml；
ESBL escherichia coli, the acetic acid ethyl ester extract of Ramulus Et Folium Pithecellobii Lucidi 10% ethanol extraction are produced in 20 strains
With amikacin associated with FIC all≤2 show that two medicines, without antagonism, wherein have 50%FIC value≤0.5
In synergism；The acetic acid ethyl ester extract of Ramulus Et Folium Pithecellobii Lucidi 10% ethanol extraction is less than or equal to 1/2MIC
Time make amikacin MIC50 be down to 4 μ g/ml from 16 alone μ g/ml, reduce 75%；MIC90
It is down to 16 μ g/ml from alone 64 μ g/ml, reduces by 75%；
20 strains are produced by the acetic acid ethyl ester extract of Ramulus Et Folium Pithecellobii Lucidi 10% ethanol extraction with bactrim combination
ESBL escherichia coli FIC all≤2 show two medicine two medicines without antagonism, wherein 20%FIC value≤0.5
In synergism.The acetic acid ethyl ester extract of Ramulus Et Folium Pithecellobii Lucidi water extract makes when less than or equal to alone MIC
Bactrim MIC50 and MIC90 is all down to 19/1 μ g/ml from 2432/128 alone μ g/ml,
Reduce 99.3%.
Embodiment 3
The acetic acid ethyl ester extract anti-product colibacillary bacteriostasis and sterilization of ESBL of Ramulus Et Folium Pithecellobii Lucidi 60% ethanol extraction
Test and respectively with amikacin, bactrim united enhanced sensitivity Effect tests.
1. experimental technique
1) mensuration of minimum inhibitory concentration (MIC):
The acetic acid ethyl ester extract of Ramulus Et Folium Pithecellobii Lucidi 60% ethanol extraction, amikacin (AMK), the new promise of compound recipe
Bright (SXT) carries out a series of doubling dilution, every hole 50 μ l, regulation respectively in MH broth bouillon
Inoculation bacterium is 1.0 × 106CFU/ml, every hole 50 μ l bacterium solution.35 DEG C of cultivations；24 hours, without being settled out
The concentration of existing minimum antibacterials is its minimum inhibitory concentration (MIC).
2) mensuration of minimum bactericidal concentration (MBC):
Use flat board coating counting method, from 1) 50ul bacteria suspension is drawn in the hole of item asepsis growth put down to blood
On plate, even spread, cultivate 24 hours for 35 DEG C, colony counting, make initial experiment viable count reduce
The concentration of 99.9% or above required minimum antibacterials is its minimum bactericidal concentration (MBC).
By measuring minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of medicine, and to data
Carry out statistics and draw MIC50, MIC90, MBC50, MBC90, evaluate medicine anti-product ESBL large intestine bar
The effect of bacterium.
3) checkerboard method:
Checkerboard method is carried out, by the acetic acid of Ramulus Et Folium Pithecellobii Lucidi 60% ethanol extraction in 96 hole aseptic culture plates
Ethyl ester extract and amikacin (AMK), bactrim (SXT) are respectively in MH broth bouillon
Doubling dilution becomes series concentration, combines respectively with two medicines each 1/4MIC to 4MIC, every hole A medicine
B medicine respectively adds 25 μ L, and the concentration adjusting bacteria suspension is 1.0 × 106CFU/ml, every hole inoculates 50 μ L bacterium solution,
35 DEG C hatch observe the associating of A medicine B medicine after 24h after to producing the colibacillary minimum inhibitory concentration of ESBLs
(MIC)。
The calculating of FIC index:Antibacterial by calculating section
Index (FIC) is evaluated the acetic acid ethyl ester extract of Ramulus Et Folium Pithecellobii Lucidi 60% ethanol extraction and is resisted with Antibiotic combination
The interaction of bacterium, FIC≤0.5 is synergism, and 0.5 FIC≤1 is summation action, 1 FIC≤2
For unrelated effect, FIC 2 is antagonism；And find out Ramulus Et Folium Pithecellobii Lucidi 60% ethanol according to asepsis growth hole and carry
Take the acetic acid ethyl ester extract of thing and the best concentration ratio of antibiotic, final evaluation Ramulus Et Folium Pithecellobii Lucidi 60% ethanol
The acetic acid ethyl ester extract of extract strengthens the effect of antibiotic effect.
2. experimental result
The acetic acid ethyl ester extract of Ramulus Et Folium Pithecellobii Lucidi 60% ethanol extraction and two kinds of antibiotic are (amikacin, multiple
Side's sulfamethoxazole) the product colibacillary extracorporeal bacteria inhibitor test of ESBL be the results are shown in Table 19.
To producing, ESBLs is colibacillary external to be killed the acetic acid ethyl ester extract of Ramulus Et Folium Pithecellobii Lucidi 60% ethanol extraction
Bacterium result of the test is shown in Table 20.
Through statistical analysis, the acetic acid ethyl ester extract of Ramulus Et Folium Pithecellobii Lucidi 60% ethanol extraction and two kinds of antibiotic
MIC50, the MIC90 producing the colibacillary In Vitro Bacteriostasis of ESBL and sterilization is shown in Table 21.
Through statistical analysis, Ramulus Et Folium Pithecellobii Lucidi extract MBC50, MBC90 to producing the colibacillary sterilization of ESBL
It is shown in Table 22.
The acetic acid ethyl ester extract of Ramulus Et Folium Pithecellobii Lucidi 60% ethanol extraction and the Combination susceptibility testing of two kinds of antibiotic
FIC value and the distribution statistics of FIC value the results are shown in Table 23, table 24.
The acetic acid ethyl ester extract of Ramulus Et Folium Pithecellobii Lucidi 60% ethanol extraction is to the sensitization of two kinds of antibiotic and connection
It is shown in Table 25-27 with rear its MIC50, MIC90.
Table 19
Table 20
Table 21
Table 22
Table 23
Table 24
Table 25
Table 26
Table 27
Test result indicate that the acetic acid ethyl ester extract of Ramulus Et Folium Pithecellobii Lucidi 60% ethanol extraction is alone to producing ESBL
Colibacillary MIC50 is 800 μ g/ml, and MIC90 is 800 μ g/ml, and MBC50 is 1600 μ g/ml,
MBC90 is 1600 μ g/ml；
ESBL escherichia coli, the acetic acid ethyl ester extract of Ramulus Et Folium Pithecellobii Lucidi 60% ethanol extraction are produced in 20 strains
With amikacin associated with FIC all≤2 show that two medicines, without antagonism, wherein have 70%FIC value≤0.5
In synergism；The acetic acid ethyl ester extract of Ramulus Et Folium Pithecellobii Lucidi 60% ethanol extraction is less than or equal to 1/4MIC
Time make amikacin MIC50 be down to 4 μ g/ml from 16 alone μ g/ml, reduce 75%；MIC90
It is down to 8 μ g/ml from alone 64 μ g/ml；
20 strains are produced by the acetic acid ethyl ester extract of Ramulus Et Folium Pithecellobii Lucidi 60% ethanol extraction with bactrim combination
ESBL escherichia coli FIC all≤2 show two medicine two medicines without antagonism, wherein 30%FIC value≤0.5
In synergism.The acetic acid ethyl ester extract of Ramulus Et Folium Pithecellobii Lucidi 60% ethanol extraction is less than or equal to alone MIC
Time make bactrim MIC50 and MIC90 all be down to from 2432/128 alone μ g/ml
19/1 μ g/ml, reduces 99.3%.
Embodiment 4
The acetic acid ethyl ester extract anti-product colibacillary bacteriostasis and sterilization of ESBL of Ramulus Et Folium Pithecellobii Lucidi 95% ethanol extraction
Test and respectively with amikacin, bactrim united enhanced sensitivity Effect tests.
1. experimental technique
1) mensuration of minimum inhibitory concentration (MIC):
The acetic acid ethyl ester extract of Ramulus Et Folium Pithecellobii Lucidi 95% ethanol extraction, amikacin (AMK), the new promise of compound recipe
Bright (SXT) carries out a series of doubling dilution, every hole 50 μ l, regulation respectively in MH broth bouillon
Inoculation bacterium is 1.0 × 106CFU/ml, every hole 50 μ l bacterium solution.35 DEG C of cultivations；24 hours, without being settled out
The concentration of existing minimum antibacterials is its minimum inhibitory concentration (MIC).
2) mensuration of minimum bactericidal concentration (MBC):
Use flat board coating counting method, from 1) 50ul bacteria suspension is drawn in the hole of item asepsis growth put down to blood
On plate, even spread, cultivate 24 hours for 35 DEG C, colony counting, make initial experiment viable count reduce
The concentration of 99.9% or above required minimum antibacterials is its minimum bactericidal concentration (MBC).
By measuring minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of medicine, and to data
Carry out statistics and draw MIC50, MIC90, MBC50, MBC90, evaluate medicine anti-product ESBL large intestine bar
The effect of bacterium.
3) checkerboard method:
Checkerboard method is carried out, by the acetic acid of Ramulus Et Folium Pithecellobii Lucidi 95% ethanol extraction in 96 hole aseptic culture plates
Ethyl ester extract and amikacin (AMK), bactrim (SXT) are respectively in MH broth bouillon
Doubling dilution becomes series concentration, combines respectively with two medicines each 1/4MIC to 4MIC, every hole A medicine
B medicine respectively adds 25 μ L, and the concentration adjusting bacteria suspension is 1.0 × 106CFU/ml, every hole inoculates 50 μ L bacterium solution,
35 DEG C hatch observe the associating of A medicine B medicine after 24h after to producing the colibacillary minimum inhibitory concentration of ESBLs
(MIC)。
The calculating of FIC index:Antibacterial by calculating section
Index (FIC) is evaluated the acetic acid ethyl ester extract of Ramulus Et Folium Pithecellobii Lucidi 95% ethanol extraction and is resisted with Antibiotic combination
The interaction of bacterium, FIC≤0.5 is synergism, and 0.5 FIC≤1 is summation action, 1 FIC≤2
For unrelated effect, FIC 2 is antagonism；And find out Ramulus Et Folium Pithecellobii Lucidi 95% ethanol according to asepsis growth hole and carry
Take the acetic acid ethyl ester extract of thing and the best concentration ratio of antibiotic, final evaluation Ramulus Et Folium Pithecellobii Lucidi 95% ethanol
The acetic acid ethyl ester extract of extract strengthens the effect of antibiotic effect.
2. experimental result
The acetic acid ethyl ester extract of Ramulus Et Folium Pithecellobii Lucidi 95% ethanol extraction and two kinds of antibiotic are (amikacin, multiple
Side's sulfamethoxazole) the product colibacillary extracorporeal bacteria inhibitor test of ESBL be the results are shown in Table 28.
To producing, ESBLs is colibacillary external to be killed the acetic acid ethyl ester extract of Ramulus Et Folium Pithecellobii Lucidi 95% ethanol extraction
Bacterium result of the test is shown in Table 29.
Through statistical analysis, the acetic acid ethyl ester extract of Ramulus Et Folium Pithecellobii Lucidi 95% ethanol extraction and two kinds of antibiotic
MIC50, the MIC90 producing the colibacillary In Vitro Bacteriostasis of ESBL and sterilization is shown in Table 30.
Through statistical analysis, Ramulus Et Folium Pithecellobii Lucidi extract MBC50, MBC90 to producing the colibacillary sterilization of ESBL
It is shown in Table 31.
The acetic acid ethyl ester extract of Ramulus Et Folium Pithecellobii Lucidi 95% ethanol extraction and the Combination susceptibility testing of two kinds of antibiotic
FIC value and the distribution statistics of FIC value the results are shown in Table 32, table 33.
The acetic acid ethyl ester extract of Ramulus Et Folium Pithecellobii Lucidi 95% ethanol extraction is to the sensitization of two kinds of antibiotic and connection
It is shown in Table 34-36 with rear its MIC50, MIC90.
Table 28
Table 29
Table 30
Table 31
Table 32
Table 33
Table 34
Table 35
Table 36
Test result indicate that the acetic acid ethyl ester extract of Ramulus Et Folium Pithecellobii Lucidi 95% ethanol extraction is alone to producing ESBL
Colibacillary MIC50 is 1600 μ g/ml, and MIC90 is 1600 μ g/ml, and MBC50 is
3200 μ g/ml, MBC90 are 3200 μ g/ml；
ESBL escherichia coli, the acetic acid ethyl ester extract of Ramulus Et Folium Pithecellobii Lucidi 95% ethanol extraction are produced in 20 strains
With amikacin associated with FIC all≤2 show that two medicines, without antagonism, wherein have 50%FIC value≤0.5
In synergism；The acetic acid ethyl ester extract of Ramulus Et Folium Pithecellobii Lucidi 95% ethanol extraction is less than or equal to 1/2MIC
Time make amikacin MIC50 be down to 4 μ g/ml from 16 alone μ g/ml, reduce 75%；MIC90
It is down to 16 μ g/ml from alone 64 μ g/ml, reduces by 75%；
20 strains are produced ESBL with bactrim combination by the acetic acid ethyl ester extract of Ramulus Et Folium Pithecellobii Lucidi 95% ethanol extraction
Escherichia coli FIC all≤2 show two medicine two medicines without antagonism, wherein 20%FIC value≤0.5 is in association
Same-action.The acetic acid ethyl ester extract of Ramulus Et Folium Pithecellobii Lucidi 95% ethanol extraction is when less than or equal to alone MIC
Bactrim MIC50 and MIC90 is made all to be down to 19/1 μ g/ml from 2432/128 alone μ g/ml,
Reduce 99.3%.
Claims (7)
1. Ramulus Et Folium Pithecellobii Lucidi extract is in preparing anti-ESBLs-producing bacteria escherichia coli medicine
Application.
2. Ramulus Et Folium Pithecellobii Lucidi extract is preparing antibiotic anti-ESBLs-producing bacteria escherichia coli
Application in hypersitization medicine.
Apply the most as claimed in claim 2, it is characterised in that described antibiotic is Ah rice
Card star or bactrim.
4. the application as described in claim 1 or 2 or 3, it is characterised in that described monkey ear
Ring extract is Ramulus Et Folium Pithecellobii Lucidi water extract or Ramulus Et Folium Pithecellobii Lucidi ethanol extraction.
Apply the most as claimed in claim 4, it is characterised in that described Ramulus Et Folium Pithecellobii Lucidi water carries
Take thing water or Ramulus Et Folium Pithecellobii Lucidi ethanol extraction is prepared by following methods: Ramulus Et Folium Pithecellobii Lucidi coarse powder water or ethanol
Extraction with aqueous solution, the extracting solution of gained is extracted with ethyl acetate again, and the extract of gained is mesh
Mark product.
Apply the most as claimed in claim 5, it is characterised in that described ethanol water
It is that meter concentration is the ethanol water of 10%-95% by volume.
Apply the most as claimed in claim 6, it is characterised in that described ethanol water
It is that meter concentration is the ethanol water of 60% by volume.
Priority Applications (9)
| Application Number | Priority Date | Filing Date | Title |
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| CN201610308718.6A CN105998153B (en) | 2016-05-10 | 2016-05-10 | Ramulus Et Folium Pithecellobii Lucidi extract is preparing the application in anti-ESBLs-producing bacteria Escherichia coli drug |
| PCT/CN2017/071671 WO2017193635A1 (en) | 2016-05-10 | 2017-01-19 | Pithecellobium clypearia benth. extract and application for preparing anti-microbial agent |
| EP17795261.1A EP3456335B1 (en) | 2016-05-10 | 2017-01-19 | Pithecellobium clypearia benth extract and application for preparing anti-microbial agent |
| US15/920,480 US11154582B2 (en) | 2016-05-10 | 2018-03-14 | Method of chinese herbal medicine extract used for treating multiple diseases caused by drug resistant bacteria infection |
| US17/129,885 US20210106640A1 (en) | 2016-05-10 | 2020-12-21 | Method of chinese herbal medicine extract used for treating multiple diseases caused by drug resistant bacteria infection |
| US17/129,901 US20210106641A1 (en) | 2016-05-10 | 2020-12-21 | Method of chinese herbal medicine extract used for treating multiple diseases caused by drug resistant bacteria infection |
| US17/129,853 US11491198B2 (en) | 2016-05-10 | 2020-12-21 | Method of chinese herbal medicine extract used for treating multiple diseases caused by drug resistant bacteria infection |
| US17/494,858 US11793849B2 (en) | 2016-05-10 | 2021-10-06 | Method of Chinese herbal medicine extract used for treating multiple diseases caused by drug resistant bacteria infection |
| US17/494,850 US11654174B2 (en) | 2016-05-10 | 2021-10-06 | Method of chinese herbal medicine extract used for treating multiple diseases caused by drug resistant bacteria infection |
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| WO2017193635A1 (en) * | 2016-05-10 | 2017-11-16 | 中山大学 | Pithecellobium clypearia benth. extract and application for preparing anti-microbial agent |
| CN107812033A (en) * | 2017-12-07 | 2018-03-20 | 成都乾坤动物药业股份有限公司 | A kind of Chewable Tablets of Chinese traditional medicine for treating pet disease of digestive tract and its production and use |
| CN115887517A (en) * | 2023-02-20 | 2023-04-04 | 云南中医药大学 | Application of laggera pterodonta extract in resisting drug-resistant bacteria producing extended-spectrum beta-lactamase |
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