CN103385912B - Pithecellobium clypearia extracts and application of extract in preparation of medicines for treating methicillin-resistant staphylococcus aureus - Google Patents
Pithecellobium clypearia extracts and application of extract in preparation of medicines for treating methicillin-resistant staphylococcus aureus Download PDFInfo
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Abstract
The invention discloses water, ethanol and ethanol aqueous extracts of traditional Chinese medicine pithecellobium clypearia and application of corresponding petroleum ether, ethyl acetate, normal butanol and a water extractants in preparation of medicines for treating methicillin-resistant staphylococcus aureus (MRSA) and antibiotics anti-MRSA sensitization medicines. Meanwhile, the invention further discloses a preparation method of the extracts or extractants. Experimental results show that water, 10% ethanol, 30% ethanol, 60% ethanol, 95% ethanol extracts of pithecellobium clypearia and corresponding ethyl acetate, normal butanol and water extractants have stronger anti-MRSA effect, wherein the activity of the ethyl acetate extracting part of the 60% ethanol extract of pithecellobium clypearia is the strongest, and the ethyl acetate extracting part of the 60% ethanol extract of pithecellobium clypearia has sensitization effect on erythrocin, ceftriaxone sodium, levofloxacin for treating MRSA.
Description
Technical field
The invention belongs to field of traditional Chinese medicine pharmacy, relate to the novelty teabag of Chinese herbal medicine Ramulus Et Folium Pithecellobii Lucidi (Pithecellobium clypearia Benth.) extract.
Background technology
As everyone knows, current bacterial resistance has become the great public health problem of global range, and antibiotic abuse is the major reason causing bacterial resistance.Methicillin-resistant staphylococcus aureus (Methicillin-resistant Staphylococcus aureus, hereinafter referred to as " MRSA ") there is multi-drug resistant and with high incidence and high mortality, it is the important pathogenic bacteria causing the diseases such as necrotizing pneumonia, Severe sepsis, necrotizing fasciitis, be one of important pathogen of hospital infection and Nosocomial Infections, bring great difficulty to clinical treatment.MRSA is to the antimicrobial drug drug resistance of the overwhelming majority, even also start to occur drug resistance to the most effective vancomycin of current clinical antimicrobial agent, once MRSA develops into the degree to the general drug resistance of vancomycin, the patient infecting MRSA will face the desperate situation pasted medical help.Therefore, the situation that directed toward bacteria drug resistance is on the rise, searching, exploitation have antimicrobial agent MRSA, antibiotic are had to the antibacterials of sensitization, very urgent.
Ramulus Et Folium Pithecellobii Lucidi is dry sprout and the leaf of Mimosaceae Ramulus Et Folium Pithecellobii Lucidi platymiscium Ramulus Et Folium Pithecellobii Lucidi (Pithecellobium clypearia Benth.), be the Chinese herbal medicine that area, Guangdong and Guangxi Provinces is commonly used, be used for the treatment of the diseases such as upper respiratory tract infection, acute pharyngolaryngitis, bacillary dysentery.There are the open water of Ramulus Et Folium Pithecellobii Lucidi of document and the simple extracting solution of ethanol to have antivirus action at present, but the medicinal application of extract prepared by other purposes or the additive method of said extracted liquid is not then reported.
Summary of the invention
The object of the invention is the novelty teabag of exploitation Ramulus Et Folium Pithecellobii Lucidi on pharmaceutical field.
One of object is, by Ramulus Et Folium Pithecellobii Lucidi water or ethanol or ethanol water solution extract for the preparation of methicillin-resistant staphylococcus aureus resistance medicine and antibiotic methicillin-resistant staphylococcus aureus resistance hypersitization medicine.
Two of object is, optimizes further Ramulus Et Folium Pithecellobii Lucidi water or ethanol or ethanol water solution extract, the active stronger extract of exploitation.
Three of object is, the preparation method of open said extracted thing.
Technical scheme of the present invention:
One, the preparation method of Ramulus Et Folium Pithecellobii Lucidi extract of the present invention, comprises the following steps:
1. get pithecellobium clypearia, extract with water or ethanol or ethanol water, recycling design obtains extractum, obtained Ramulus Et Folium Pithecellobii Lucidi water extract or ethanol extraction or ethanol water solution extract.
2. above-mentioned extractum thin up, then with petroleum ether or ethyl acetate or n-butanol extraction, get extract and concentrate obtained corresponding Ramulus Et Folium Pithecellobii Lucidi extract respectively.The composition of this extract is optimized further, and its drug effect significantly improves.The unit of the medical material weight related in preparation and solvent volume ratio, medical material weight in gram, solvent volume is in milliliter.
Two, above-mentioned Ramulus Et Folium Pithecellobii Lucidi extract is applied to the preparation of methicillin-resistant staphylococcus aureus resistance medicine as active component.
Three, above-mentioned Ramulus Et Folium Pithecellobii Lucidi extract is applied to the preparation of antibiotic methicillin-resistant staphylococcus aureus resistance hypersitization medicine as active component.Particularly as the sensitizer of erythromycin, ceftriaxone sodium, the anti-MRSA composition of medicine of levofloxacin.
The present invention is by carrying out Extraction and separation to Ramulus Et Folium Pithecellobii Lucidi, the pharmacological action of each extract methicillin-resistant staphylococcus aureus resistance (MRSA) is screened, and carry out Combination susceptibility testing, investigate the anti-MRSA of Ramulus Et Folium Pithecellobii Lucidi and the sensitization to the anti-MRSA of antibiotic thereof.Experimental result shows: Ramulus Et Folium Pithecellobii Lucidi water, 10% ethanol, 30% ethanol, 60% ethanol, 95% ethanol extraction and corresponding ethyl acetate, n-butyl alcohol and water layer extract all have stronger anti-MRSA effect, wherein the acetic acid ethyl ester extract activity of Ramulus Et Folium Pithecellobii Lucidi 60% ethanol extraction is the strongest, strong 4 times relative to the antibacterial action of the water extract without extraction; The acetic acid ethyl ester extract of Ramulus Et Folium Pithecellobii Lucidi 60% ethanol extraction has sensitization to erythromycin, ceftriaxone sodium, the anti-MRSA of levofloxacin.
The present invention is screened by the pharmacological action of following method to the methicillin-resistant staphylococcus aureus resistance of Ramulus Et Folium Pithecellobii Lucidi extract.
With micro-broth dilution method Ramulus Et Folium Pithecellobii Lucidi extract to the minimum inhibitory concentration (MIC) of methicillin-resistant staphylococcus aureus and minimum bactericidal concentration (MBC).
Bacterial strain: methicillin-resistant staphylococcus aureus (MRSA is numbered M1-M20) clinical separation strain 20 strain; Staphylococcus aureus reference culture (Sa, ATCC25923) provides by clinical microbiology laboratory technique room, laboratory medicine portion of No.1 Hospital Affiliated to Zhongshan Univ., detects confirm its drug resistance through No.1 Hospital Affiliated to Zhongshan Univ.'s clinical microbiology laboratory technique room.
MH broth bouillon: MH meat soup dry powder (Britain OXOID LTD.) 2.1g, be settled to 100ml, NaOH regulates pH to 7.0, autoclaving, puts 4 DEG C of refrigerators for subsequent use.
Method: the micro-broth dilution method operation recommended with reference to US National clinical trial standardization committee (NCCLS).
The present invention is investigated the sensitization of Ramulus Et Folium Pithecellobii Lucidi extract to erythromycin, ceftriaxone sodium, levofloxacin methicillin-resistant staphylococcus aureus resistance by following method.
Bacterial strain: methicillin-resistant staphylococcus aureus (MRSA is numbered M1-M20) clinical separation strain 100 strain; Staphylococcus aureus reference culture (Sa, ATCC25923) provides by clinical microbiology laboratory technique room, laboratory medicine portion of No.1 Hospital Affiliated to Zhongshan Univ., detects confirm its drug resistance through No.1 Hospital Affiliated to Zhongshan Univ.'s clinical microbiology laboratory technique room.
MH broth bouillon: MH meat soup dry powder (Britain OXOID LTD.) 2.1g, be settled to 100ml, NaOH regulates pH to 7.0, autoclaving, puts 4 DEG C of refrigerators for subsequent use.
Method: the checkerboard method operation recommended with reference to US National clinical trial standardization committee (NCCLS).
The invention has the beneficial effects as follows:
There is with the product that the inventive method prepares the effect of methicillin-resistant staphylococcus aureus resistance (MRSA), and to erythromycin, ceftriaxone sodium, the anti-MRSA of levofloxacin, there is sensitization, can be used as natural antibacterial, antibiotic sensitizer, be applied to the industry such as food, medicine; Realize the comprehensive utilization of Ramulus Et Folium Pithecellobii Lucidi plant simultaneously and improve its added value.Medicine alleged by the present invention comprises people's medicine and other medicine for animal.
Above-mentioned Ramulus Et Folium Pithecellobii Lucidi extract of the present invention can be used as the active component of antimicrobial DP finish preparation, makes tablet, capsule, slow releasing agent, injection, suppository, compound preparation etc.
Detailed description of the invention
Technical scheme of the present invention is further illustrated below by way of specific embodiment.
Embodiment 1
Prepare Ramulus Et Folium Pithecellobii Lucidi water extract and extract thereof
Ramulus Et Folium Pithecellobii Lucidi (Pithecellobium clypearia Benth.) is provided by Guangzhou Huacheng pharmaceutical plant.Ramulus Et Folium Pithecellobii Lucidi 300g, decocts 2 times with water 3000ml, each 2 hours, filters; Merging filtrate, concentrates to obtain extractum (water extract) 81.9g.Get extractum 54.6g, after water 200ml suspendible, with each 200ml, use petroleum ether, ethyl acetate, n-butanol extraction respectively, often kind of solvent extraction three times, merge each extract respectively, concentrate to obtain ligroin extraction 0.9g, ethyl acetate extract 14.8g, n-butanol extract 11.7g, water layer extract 27.1g.
Embodiment 2
The screening of the pharmacological action of Ramulus Et Folium Pithecellobii Lucidi water extract and extract methicillin-resistant staphylococcus aureus resistance (MRSA) thereof
1 experimental technique
1) mensuration of minimum inhibitory concentration (MIC):
Ramulus Et Folium Pithecellobii Lucidi water extract and extract thereof, erythromycin, ceftriaxone sodium, levofloxacin, vancomycin carry out a series of doubling dilution respectively in MH broth bouillon, regulate inoculation bacterium to be 1.0 × 10
5cFU/ml.Cultivate 24 hours for 35 DEG C, the concentration without the minimum antibacterials of precipitation appearance is its minimum inhibitory concentration (MIC).
2) mensuration of minimum bactericidal concentration (MBC):
From 1) pipette 50 μ l on blood plate the hole of item asepsis growth, even spread, cultivate 24 hours for 35 DEG C, colony counting, the concentration of the minimum antibacterials making initial experiment viable count minimizing 99.9% or more required is its minimum bactericidal concentration (MBC).
By measuring the minimum inhibitory concentration (MIC) of medicine and minimum bactericidal concentration (MBC), and statistics is carried out to data draw MIC
50, MIC
90, MBC
50, MBC
90, evaluate the effect of medicine methicillin-resistant staphylococcus aureus resistance.
2 experimental results
Ramulus Et Folium Pithecellobii Lucidi water extract and extract thereof, four kinds of antibiotic (erythromycin, ceftriaxone sodium, levofloxacin, vancomycin) the results are shown in Table 1 to the extracorporeal bacteria inhibitor test of methicillin-resistant staphylococcus aureus.
Ramulus Et Folium Pithecellobii Lucidi water extract and extract thereof to the extracorporeal disinfecting result of the test of methicillin-resistant staphylococcus aureus in table 2.
Through statistical analysis, Ramulus Et Folium Pithecellobii Lucidi water extract and the In Vitro Bacteriostasis of extract to methicillin-resistant staphylococcus aureus thereof and the MIC of bactericidal assay
50, MIC
90, MBC
50, MBC
90in table 3.
Through statistical analysis, four kinds of antibiotic (erythromycin, ceftriaxone sodium, levofloxacin, vancomycin) MIC to the bacteriostatic test of methicillin-resistant staphylococcus aureus
50, MIC
90in table 4:
Table 1 Ramulus Et Folium Pithecellobii Lucidi water extract and extract thereof, four kinds of antibiotic are to the extracorporeal bacteria inhibitor test result of MRSA
Table 2 Ramulus Et Folium Pithecellobii Lucidi water extract and extract thereof are to the extracorporeal disinfecting result of the test of MRSA
Table 3 Ramulus Et Folium Pithecellobii Lucidi water extract and the In Vitro Bacteriostasis of extract to MRSA thereof and the statistical result of bactericidal assay
Table 4 four kinds of antibiotic are to the statistical result of the extracorporeal bacteria inhibitor test of MRSA
Experimental result shows, Ramulus Et Folium Pithecellobii Lucidi water extract and acetic acid ethyl ester extract thereof, n-butyl alcohol extract, water layer have methicillin-resistant staphylococcus aureus resistance (MRSA) effect.Wherein the activity of the anti-MRSA of the acetic acid ethyl ester extract of water extract is the strongest.
Embodiment 3
Prepare Ramulus Et Folium Pithecellobii Lucidi 10% ethanol extraction and extract thereof
Ramulus Et Folium Pithecellobii Lucidi (Pithecellobium clypearia Benth.) is provided by Guangzhou Huacheng pharmaceutical plant.Ramulus Et Folium Pithecellobii Lucidi 300g, by 10% ethanol 3000ml reflux, extract, 2 times, each 2 hours, filters; Merging filtrate, concentrates to obtain extractum (10% ethanol extraction) 95.1g.Get extractum 63.4g, use petroleum ether, ethyl acetate, n-butanol extraction respectively, often kind of solvent extraction three times, each 200ml, merge each extract respectively, concentrate to obtain petroleum ether extract 1.2g, acetic acid ethyl ester extract 17.3g, n-butyl alcohol extract 16.3g, water layer extract 28.6g.
Embodiment 4
The screening of the pharmacological action of Ramulus Et Folium Pithecellobii Lucidi 10% ethanol extraction and extract methicillin-resistant staphylococcus aureus resistance (MRSA) thereof
1 experimental technique
3) mensuration of minimum inhibitory concentration (MIC):
Ramulus Et Folium Pithecellobii Lucidi 10% ethanol extraction and extract thereof, erythromycin, ceftriaxone sodium, levofloxacin, vancomycin carry out a series of doubling dilution respectively in MH broth bouillon, regulate inoculation bacterium to be 1.0 × 10
5cFU/ml.Cultivate 24 hours for 35 DEG C, the concentration without the minimum antibacterials of precipitation appearance is its minimum inhibitory concentration (MIC).
4) mensuration of minimum bactericidal concentration (MBC):
From 1) pipette 50 μ l on blood plate the hole of item asepsis growth, even spread, cultivate 24 hours for 35 DEG C, colony counting, the concentration of the minimum antibacterials making initial experiment viable count minimizing 99.9% or more required is its minimum bactericidal concentration (MBC).
By measuring the minimum inhibitory concentration (MIC) of medicine and minimum bactericidal concentration (MBC), and statistics is carried out to data draw MIC
50, MIC
90, MBC
50, MBC
90, evaluate the effect of medicine methicillin-resistant staphylococcus aureus resistance.
2 experimental results
Ramulus Et Folium Pithecellobii Lucidi 10% ethanol extraction and extract thereof, four kinds of antibiotic (erythromycin, ceftriaxone sodium, levofloxacin, vancomycin) the results are shown in Table 5 to the extracorporeal bacteria inhibitor test of methicillin-resistant staphylococcus aureus.
Ramulus Et Folium Pithecellobii Lucidi 10% ethanol extraction and extract thereof to the extracorporeal disinfecting result of the test of methicillin-resistant staphylococcus aureus in table 6.
Through statistical analysis, Ramulus Et Folium Pithecellobii Lucidi 10% ethanol extraction and the In Vitro Bacteriostasis of extract to methicillin-resistant staphylococcus aureus thereof and the MIC of bactericidal assay
50, MIC
90, MBC
50, MBC
90in table 7.
Through statistical analysis, four kinds of antibiotic (erythromycin, ceftriaxone sodium, levofloxacin, vancomycin) MIC to the bacteriostatic test of methicillin-resistant staphylococcus aureus
50, MIC
90in table 8:
Table 5 Ramulus Et Folium Pithecellobii Lucidi 10% ethanol extraction and extract thereof, four kinds of antibiotic are to the extracorporeal bacteria inhibitor test result of MRSA
Table 6 Ramulus Et Folium Pithecellobii Lucidi 10% ethanol extraction and extract thereof are to the extracorporeal disinfecting result of the test of MRSA
Table 7 Ramulus Et Folium Pithecellobii Lucidi 10% ethanol extraction and the In Vitro Bacteriostasis of extract to MRSA thereof and the statistical result of bactericidal assay
Table 8 four kinds of antibiotic are to the statistical result of the extracorporeal bacteria inhibitor test of MRSA
Experimental result shows, Ramulus Et Folium Pithecellobii Lucidi 10% ethanol extraction and acetic acid ethyl ester extract thereof, n-butyl alcohol extract, water layer have methicillin-resistant staphylococcus aureus resistance (MRSA) effect.Wherein the activity of the anti-MRSA of the acetic acid ethyl ester extract of 10% ethanol extraction is the strongest.
Embodiment 5
Prepare Ramulus Et Folium Pithecellobii Lucidi 30% ethanol extraction and extract thereof
Ramulus Et Folium Pithecellobii Lucidi (Pithecellobium clypearia Benth.) is provided by Guangzhou Huacheng pharmaceutical plant.Ramulus Et Folium Pithecellobii Lucidi 300g, by 30% ethanol 3000ml reflux, extract, 2 times, each 2 hours, filters; Merging filtrate, concentrates to obtain extractum (30% ethanol extraction) 115.5g.Get extractum 77.0g, use petroleum ether, ethyl acetate, n-butanol extraction respectively, often kind of solvent extraction three times, each 200ml, merge each extract respectively, concentrate to obtain petroleum ether extract 1.6g, acetic acid ethyl ester extract 21.7g, n-butyl alcohol extract 17.6g, water layer extract 36.1g.
Embodiment 6
The screening of the pharmacological action of Ramulus Et Folium Pithecellobii Lucidi 30% ethanol extraction and extract methicillin-resistant staphylococcus aureus resistance (MRSA) thereof
1 experimental technique
5) mensuration of minimum inhibitory concentration (MIC):
Ramulus Et Folium Pithecellobii Lucidi 30% ethanol extraction and extract thereof, erythromycin, ceftriaxone sodium, levofloxacin, vancomycin carry out a series of doubling dilution respectively in MH broth bouillon, regulate inoculation bacterium to be 1.0 × 10
5cFU/ml.Cultivate 24 hours for 35 DEG C, the concentration without the minimum antibacterials of precipitation appearance is its minimum inhibitory concentration (MIC).
6) mensuration of minimum bactericidal concentration (MBC):
From 1) pipette 50 μ l on blood plate the hole of item asepsis growth, even spread, cultivate 24 hours for 35 DEG C, colony counting, the concentration of the minimum antibacterials making initial experiment viable count minimizing 99.9% or more required is its minimum bactericidal concentration (MBC).
By measuring the minimum inhibitory concentration (MIC) of medicine and minimum bactericidal concentration (MBC), and statistics is carried out to data draw MIC
50, MIC
90, MBC
50, MBC
90, evaluate the effect of medicine methicillin-resistant staphylococcus aureus resistance.
2 experimental results
Ramulus Et Folium Pithecellobii Lucidi 30% ethanol extraction and extract thereof, four kinds of antibiotic (erythromycin, ceftriaxone sodium, levofloxacin, vancomycin) the results are shown in Table 9 to the extracorporeal bacteria inhibitor test of methicillin-resistant staphylococcus aureus.
Ramulus Et Folium Pithecellobii Lucidi 30% ethanol extraction and extract thereof to the extracorporeal disinfecting result of the test of methicillin-resistant staphylococcus aureus in table 10.
Through statistical analysis, Ramulus Et Folium Pithecellobii Lucidi 30% ethanol extraction and the In Vitro Bacteriostasis of extract to methicillin-resistant staphylococcus aureus thereof and the MIC of bactericidal assay
50, MIC
90, MBC
50, MBC
90in table 11.
Through statistical analysis, four kinds of antibiotic (erythromycin, ceftriaxone sodium, levofloxacin, vancomycin) MIC to the bacteriostatic test of methicillin-resistant staphylococcus aureus
50, MIC
90in table 12:
Table 9 Ramulus Et Folium Pithecellobii Lucidi 30% ethanol extraction and extract thereof, four kinds of antibiotic are to the extracorporeal bacteria inhibitor test result of MRSA
Table 10 Ramulus Et Folium Pithecellobii Lucidi 30% ethanol extraction and extract thereof are to the extracorporeal disinfecting result of the test of MRSA
Table 11 Ramulus Et Folium Pithecellobii Lucidi 30% ethanol extraction and the In Vitro Bacteriostasis of extract to MRSA thereof and the statistical result of bactericidal assay
Table 12 four kinds of antibiotic are to the statistical result of the extracorporeal bacteria inhibitor test of MRSA
Experimental result shows, Ramulus Et Folium Pithecellobii Lucidi 30% ethanol extraction and acetic acid ethyl ester extract thereof, n-butyl alcohol extract, water layer have methicillin-resistant staphylococcus aureus resistance (MRSA) effect.Wherein the activity of the anti-MRSA of the acetic acid ethyl ester extract of 30% ethanol extraction is the strongest.
Embodiment 7
Prepare Ramulus Et Folium Pithecellobii Lucidi 95% ethanol extraction and extract thereof
Ramulus Et Folium Pithecellobii Lucidi (Pithecellobium clypearia Benth.) is provided by Guangzhou Huacheng pharmaceutical plant.Ramulus Et Folium Pithecellobii Lucidi 300g, by 95% ethanol 3000ml reflux, extract, 2 times, each 2 hours, filters; Merging filtrate, concentrates to obtain extractum (95% ethanol extraction) 119.4g.Get extractum 79.6g, use petroleum ether, ethyl acetate, n-butanol extraction respectively, often kind of solvent extraction three times, each 200ml, merge each extract respectively, concentrate to obtain petroleum ether extract 2.3g, acetic acid ethyl ester extract 22.5g, n-butyl alcohol extract 23.3g, water layer extract 31.4g.
Embodiment 8
The screening of the pharmacological action of Ramulus Et Folium Pithecellobii Lucidi 95% ethanol extraction and extract methicillin-resistant staphylococcus aureus resistance (MRSA) thereof
1 experimental technique
7) mensuration of minimum inhibitory concentration (MIC):
Ramulus Et Folium Pithecellobii Lucidi 95% ethanol extraction and extract thereof, erythromycin, ceftriaxone sodium, levofloxacin, vancomycin carry out a series of doubling dilution respectively in MH broth bouillon, regulate inoculation bacterium to be 1.0 × 10
5cFU/ml.Cultivate 24 hours for 35 DEG C, the concentration without the minimum antibacterials of precipitation appearance is its minimum inhibitory concentration (MIC).
8) mensuration of minimum bactericidal concentration (MBC):
From 1) pipette 50 μ l on blood plate the hole of item asepsis growth, even spread, cultivate 24 hours for 35 DEG C, colony counting, the concentration of the minimum antibacterials making initial experiment viable count minimizing 99.9% or more required is its minimum bactericidal concentration (MBC).
By measuring the minimum inhibitory concentration (MIC) of medicine and minimum bactericidal concentration (MBC), and statistics is carried out to data draw MIC
50, MIC
90, MBC
50, MBC
90, evaluate the effect of medicine methicillin-resistant staphylococcus aureus resistance.
2 experimental results
Ramulus Et Folium Pithecellobii Lucidi 95% ethanol extraction and extract thereof, four kinds of antibiotic (erythromycin, ceftriaxone sodium, levofloxacin, vancomycin) the results are shown in Table 13 to the extracorporeal bacteria inhibitor test of methicillin-resistant staphylococcus aureus.
Ramulus Et Folium Pithecellobii Lucidi 95% ethanol extraction and extract thereof to the extracorporeal disinfecting result of the test of methicillin-resistant staphylococcus aureus in table 14.
Through statistical analysis, Ramulus Et Folium Pithecellobii Lucidi 95% ethanol extraction and the In Vitro Bacteriostasis of extract to methicillin-resistant staphylococcus aureus thereof and the MIC of bactericidal assay
50, MIC
90, MBC
50, MBC
90in table 15.
Through statistical analysis, four kinds of antibiotic (erythromycin, ceftriaxone sodium, levofloxacin, vancomycin) MIC to the bacteriostatic test of methicillin-resistant staphylococcus aureus
50, MIC
90in table 16:
Table 13 Ramulus Et Folium Pithecellobii Lucidi 95% ethanol extraction and extract thereof, four kinds of antibiotic are to the extracorporeal bacteria inhibitor test result of MRSA
Table 14 Ramulus Et Folium Pithecellobii Lucidi 95% ethanol extraction and extract thereof are to the extracorporeal disinfecting result of the test of MRSA
Table 15 Ramulus Et Folium Pithecellobii Lucidi 95% ethanol extraction and the In Vitro Bacteriostasis of extract to MRSA thereof and the statistical result of bactericidal assay
Table 16 four kinds of antibiotic are to the statistical result of the extracorporeal bacteria inhibitor test of MRSA
Experimental result shows, Ramulus Et Folium Pithecellobii Lucidi 95% ethanol extraction and acetic acid ethyl ester extract thereof, n-butyl alcohol extract, water layer have methicillin-resistant staphylococcus aureus resistance (MRSA) effect.Wherein the activity of the anti-MRSA of the acetic acid ethyl ester extract of 95% ethanol extraction is the strongest.
Embodiment 9
Prepare Ramulus Et Folium Pithecellobii Lucidi 60% ethanol extraction and extract thereof
Ramulus Et Folium Pithecellobii Lucidi (Pithecellobium clypearia Benth.) is provided by Guangzhou Huacheng pharmaceutical plant.Ramulus Et Folium Pithecellobii Lucidi 300g breaks into coarse powder, refluxes 2 times with 60% ethanol 3000ml, each 2 hours, filters; Merging filtrate, concentrates to obtain extractum (60% ethanol extraction) 133.8g.Get extractum 89.2g, after water 200ml suspendible, with each 200ml, use petroleum ether, ethyl acetate, n-butanol extraction respectively, often kind of solvent extraction three times, merge each extract respectively, concentrate to obtain ligroin extraction 2.3g, ethyl acetate extract 26.2g, n-butanol extract 19.5g, water layer extract 41.1g.
Embodiment 10
The screening of the pharmacological action of Ramulus Et Folium Pithecellobii Lucidi 60% extract and extract methicillin-resistant staphylococcus aureus resistance thereof
1 experimental technique
9) mensuration of minimum inhibitory concentration (MIC):
Ramulus Et Folium Pithecellobii Lucidi extract, erythromycin, ceftriaxone sodium, levofloxacin, vancomycin carry out a series of doubling dilution respectively in MH broth bouillon, regulate inoculation bacterium to be 1.0 × 10
5cFU/ml.Cultivate 24 hours for 35 DEG C, the concentration without the minimum antibacterials of precipitation appearance is its minimum inhibitory concentration (MIC).
10) mensuration of minimum bactericidal concentration (MBC):
From 1) pipette 50 μ l on blood plate the hole of item asepsis growth, even spread, cultivate 24 hours for 35 DEG C, colony counting, the concentration of the minimum antibacterials making initial experiment viable count minimizing 99.9% or more required is its minimum bactericidal concentration (MBC).
By measuring the minimum inhibitory concentration (MIC) of medicine and minimum bactericidal concentration (MBC), and statistics is carried out to data draw MIC
50, MIC
90, MBC
50, MBC
90, evaluate the effect of medicine methicillin-resistant staphylococcus aureus resistance.
2 experimental results
Ramulus Et Folium Pithecellobii Lucidi extract and four kinds of antibiotic (erythromycin, ceftriaxone sodium, levofloxacin, vancomycin) the results are shown in Table 17 to the extracorporeal bacteria inhibitor test of methicillin-resistant staphylococcus aureus.
Ramulus Et Folium Pithecellobii Lucidi extract to the extracorporeal disinfecting result of the test of methicillin-resistant staphylococcus aureus in table 18.
Through statistical analysis, the In Vitro Bacteriostasis of Ramulus Et Folium Pithecellobii Lucidi extract to methicillin-resistant staphylococcus aureus and the MIC of bactericidal assay
50, MIC
90, MBC
50, MBC
90in table 19.
Through statistical analysis, four kinds of antibiotic (erythromycin, ceftriaxone sodium, levofloxacin, vancomycin) MIC to the bacteriostatic test of methicillin-resistant staphylococcus aureus
50, MIC
90in table 20:
Table 17 Ramulus Et Folium Pithecellobii Lucidi 60% ethanol extraction and extract thereof, four kinds of antibiotic are to the extracorporeal bacteria inhibitor test result of MRSA
Table 18 Ramulus Et Folium Pithecellobii Lucidi 60% ethanol extraction and extract thereof are to the extracorporeal disinfecting result of the test of MRSA
Table 19 Ramulus Et Folium Pithecellobii Lucidi 60% ethanol extraction and the In Vitro Bacteriostasis of extract to MRSA thereof and the statistical result of bactericidal assay
Table 20 four kinds of antibiotic are to the statistical result of the extracorporeal bacteria inhibitor test of MRSA
Experimental result shows, Ramulus Et Folium Pithecellobii Lucidi 60% ethanol extraction and acetic acid ethyl ester extract thereof, n-butyl alcohol extract, water layer have stronger methicillin-resistant staphylococcus aureus resistance (MRSA) effect.Wherein the activity of acetic acid ethyl ester extract (i.e. extract of the present invention) the anti-MRSA of 60% ethanol extraction is the strongest.
Embodiment 11
The acetic acid ethyl ester extract of Ramulus Et Folium Pithecellobii Lucidi 60% ethanol extraction is to the investigation of the sensitization of erythromycin, ceftriaxone sodium, levofloxacin methicillin-resistant staphylococcus aureus resistance
1 experimental technique
Checkerboard method:
Adopt checkerboard method, the acetic acid ethyl ester extract of Ramulus Et Folium Pithecellobii Lucidi 60% ethanol extraction, erythromycin (ERY), ceftriaxone sodium (CRO), levofloxacin (LVX) are carried out a series of doubling dilution respectively in MH broth bouillon, and the concentration of adjustment bacteria suspension is 1.0 × 10
5cFU/mL, is inoculated in 96 hole aseptic culture plates, cultivates 24 hours for 35 DEG C, observes the medicine after compatibility to the MIC of methicillin-resistant staphylococcus aureus.Concentration without the minimum antibacterials of precipitation appearance is its minimum inhibitory concentration (MIC).
According to formula
, (FIC≤0.5 represents that two kinds of medicines present synergism to evaluate Ramulus Et Folium Pithecellobii Lucidi ethyl acetate extract and the antibacterial interaction of Antibiotic combination by calculating section Mlc index (FIC); 0.5 < FIC≤1 represents that two kinds of medicines present summation action; 1 < FIC≤2 represent that two kinds of medicines are without interaction; FIC > 2 represents that two kinds of medicines present antagonism), and find out the acetic acid ethyl ester extract of Ramulus Et Folium Pithecellobii Lucidi 60% ethanol extraction and antibiotic best concentration ratio according to asepsis growth hole, the acetic acid ethyl ester extract of final evaluation Ramulus Et Folium Pithecellobii Lucidi 60% ethanol extraction is to the drug effect of antibiotic sensitization.
2 experimental results
The acetic acid ethyl ester extract of Ramulus Et Folium Pithecellobii Lucidi 60% ethanol extraction and the FIC value of Antibiotic combination drug sensitive test are in table 21.The acetic acid ethyl ester extract of Ramulus Et Folium Pithecellobii Lucidi 60% ethanol extraction and the result of erythromycin to 20 strain MRSA and a strain ATCC25923 drug combination are FIC≤0.5, in synergism; Ramulus Et Folium Pithecellobii Lucidi ethyl acetate extract and the result of ceftriaxone sodium to a strain ATCC25923 drug combination are FIC=0.5, in synergism; Ramulus Et Folium Pithecellobii Lucidi ethyl acetate extract and the result of ceftriaxone sodium to 20 strain MRSA drug combinations are 0.5 < FIC≤1, in summation action; Ramulus Et Folium Pithecellobii Lucidi ethyl acetate extract and the result of levofloxacin to 20 strain MRSA and a strain ATCC25923 drug combination are FIC=1, in summation action.
The sensitization of acetic acid ethyl ester extract to the anti-MRSA of antibiotic of Ramulus Et Folium Pithecellobii Lucidi 60% ethanol extraction the results are shown in Table 22.For M1 MRSA, the Ramulus Et Folium Pithecellobii Lucidi ethyl acetate extract of 100 μ g/ml and ceftriaxone sodium administering drug combinations, can be reduced to 256 μ g/ml (MIC during conbined usage) by the MIC of ceftriaxone from 1024 μ g/ml (MIC when being used alone).Experimental result shows, and for all strain subjects, the acetic acid ethyl ester extract of Ramulus Et Folium Pithecellobii Lucidi 60% ethanol extraction all can reduce the MIC of erythromycin, ceftriaxone sodium, levofloxacin.Illustrate that Ramulus Et Folium Pithecellobii Lucidi ethyl acetate extract has the sensitization to antibiotic methicillin-resistant staphylococcus aureus resistance
The acetic acid ethyl ester extract of table 21 Ramulus Et Folium Pithecellobii Lucidi 60% ethanol extraction and the FIC value of Antibiotic combination medication
| Strain number | EA+CRO | EA+LVX | EA+ERY | Strain number | EA+CRO | EA+LVX | EA+ERY |
| M1 | 0.75 | 1 | 0.25 | M11 | 0.75 | 1 | 0.5 |
| M2 | 0.75 | 1 | 0.25 | M12 | 0.625 | 1 | 0.5 |
| M3 | 0.75 | 1 | 0.25 | M13 | 1 | 1 | 0.5 |
| M4 | 1 | 1 | 0.5 | M14 | 1 | 1 | 0.5 |
| M5 | 1 | 1 | 0.5 | M15 | 1 | 1 | 0.5 |
| M6 | 1 | 1 | 0.5 | M16 | 1 | 1 | 0.5 |
| M7 | 1 | 1 | 0.5 | M17 | 1 | 1 | 0.5 |
| M8 | 1 | 1 | 0.5 | M18 | 1 | 1 | 0.5 |
| M9 | 1 | 1 | 0.5 | M19 | 1 | 1 | 0.375 |
| M10 | 1 | 1 | 0.5 | M20 | 0.75 | 1 | 0.375 |
| ATCC25923 | 0.5 | 0.625 | 0.5 |
The acetic acid ethyl ester extract of table 22 Ramulus Et Folium Pithecellobii Lucidi 60% ethanol extraction is to the sensitization of the anti-MRSA of antibiotic
| Strain number | EA | CRO | EA | LVX | EA | ERY |
| M1 | 100 | 1024→256 | 100 | 32→16 | 25 | 512→64 |
| M2 | 100 | 1024→256 | 100 | 32→16 | 25 | 512→64 |
| M3 | 100 | 1024→256 | 100 | 16→8 | 25 | 512→64 |
| M4 | 100 | 512→256 | 100 | 32→16 | 50 | 512→128 |
| M5 | 100 | 1024→512 | 100 | 32→16 | 50 | 512→128 |
| M6 | 100 | 1024→512 | 100 | 32→16 | 50 | 512→128 |
| M7 | 100 | 1024→512 | 100 | 32→16 | 50 | 512→128 |
| M8 | 100 | 512→256 | 100 | 8→4 | 50 | 256→64 |
| M9 | 100 | 64→32 | 100 | 8→4 | 50 | 256→64 |
| M10 | 100 | 128→64 | 100 | 4→2 | 50 | 512→128 |
| M11 | 100 | 1024→256 | 100 | 32→16 | 50 | 256→64 |
| M12 | 100 | 64→8 | 100 | 4→2 | 50 | 256→64 |
| M13 | 100 | 1024→512 | 100 | 16→8 | 50 | 256→64 |
| M14 | 100 | 64→32 | 100 | 8→4 | 50 | 64→16 |
| M15 | 100 | 1024→512 | 100 | 32→16 | 50 | 256→64 |
| M16 | 100 | 64→32 | 100 | 8→4 | 50 | 1024→256 |
| M17 | 100 | 64→32 | 100 | 4→2 | 50 | 1024→256 |
| M18 | 100 | 128→64 | 100 | 32→16 | 50 | 512→64 |
| M19 | 100 | 2560→1280 | 100 | 32→16 | 50 | 512→64 |
| M20 | 50 | 2560→1280 | 100 | 32→16 | 50 | 512→64 |
| ATCC25923 | 50 | 8→2 | 100 | 0.25→0.03 | 50 | 0.5→0.13 |
Experimental result shows, extract of the present invention and erythromycin are synergism, and itself and ceftriaxone, levofloxacin are summation action respectively.And extract of the present invention has stronger sensitization to erythromycin, ceftriaxone sodium, levofloxacin methicillin-resistant staphylococcus aureus resistance.
Claims (7)
1. Ramulus Et Folium Pithecellobii Lucidi water extract or ethanol extraction or ethanol water solution extract are preparing the application in methicillin-resistant staphylococcus aureus resistance medicine.
2. apply as claimed in claim 1, it is characterized in that: the preparation method of described extract is: get pithecellobium clypearia, extract with water or ethanol or ethanol water, recycling design extractum, extractum thin up, again with ethyl acetate or n-butanol extraction, extract concentrates to obtain Ramulus Et Folium Pithecellobii Lucidi extract.
3. apply as claimed in claim 2, it is characterized in that: the concentration of alcohol of described ethanol water is 10%, 30%, 60% or 95%.
4. apply as claimed in claim 2, it is characterized in that, described preparation method comprises the following steps: get pithecellobium clypearia, extract three times with 60% ethanol water than the ratio for 1:10 in medical material weight and solvent volume, recycling design obtains extractum, use equal-volume extraction into ethyl acetate three times after extractum thin up, combining extraction liquid is concentrated to obtain described Ramulus Et Folium Pithecellobii Lucidi extract.
5. the acetic acid ethyl ester extract of Ramulus Et Folium Pithecellobii Lucidi 60% ethanol water solution extract is preparing the application in antibiotic methicillin-resistant staphylococcus aureus resistance hypersitization medicine.
6. apply as claimed in claim 5, it is characterized in that: described extract is prepared by following methods: get pithecellobium clypearia, extract three times with 60% ethanol water than the ratio for 1:10 in medical material weight and solvent volume, recycling design obtains extractum, use equal-volume extraction into ethyl acetate three times after extractum thin up, combining extraction liquid is concentrated to obtain described Ramulus Et Folium Pithecellobii Lucidi extract.
7. apply as claimed in claim 5, it is characterized in that: described antibiotic is erythromycin or ceftriaxone sodium or levofloxacin.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104974018B (en) * | 2015-06-15 | 2016-08-24 | 沈阳药科大学 | Compound extracted from Chinese medicine Ramulus Et Folium Pithecellobii Lucidi and application thereof |
| CN105963339B (en) * | 2016-05-10 | 2019-09-17 | 中山大学 | Ramulus Et Folium Pithecellobii Lucidi extract is preparing the application in anti-multi-resistant Pseudomonas aeruginosa drug |
| CN105816511B (en) * | 2016-05-10 | 2019-09-17 | 中山大学 | Ramulus Et Folium Pithecellobii Lucidi extract is preparing the application in anti-Multi-drug resistant Acinetobacter baumannii drug |
| WO2017193635A1 (en) | 2016-05-10 | 2017-11-16 | 中山大学 | Pithecellobium clypearia benth. extract and application for preparing anti-microbial agent |
| CN105998153B (en) * | 2016-05-10 | 2019-09-17 | 中山大学 | Ramulus Et Folium Pithecellobii Lucidi extract is preparing the application in anti-ESBLs-producing bacteria Escherichia coli drug |
| CN107970272A (en) * | 2017-12-22 | 2018-05-01 | 成都乾坤动物药业股份有限公司 | The purposes of pithecellobium clypearia or extract in treatment and/or the bronchopneumonic medicine of auxiliary treatment livestock and poultry is prepared |
| CN111012819A (en) * | 2019-12-31 | 2020-04-17 | 江西杏林白马药业有限公司 | A nanometer preparation of Pithecellobium clypearia extract and its preparation method |
| CN111317791A (en) * | 2020-02-27 | 2020-06-23 | 江西杏林白马药业股份有限公司 | Heat-clearing and stranguria-treating tablet and preparation method thereof |
| CN111904995B (en) * | 2020-08-04 | 2022-03-01 | 南方医科大学 | Application of prunella vulgaris combined antibiotic in preparation of medicine for resisting multidrug-resistant staphylococcus aureus |
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