CN101940698B - Drug combination for eliminating neisseria gonorrhoeae resistance plasmids and preparation method thereof - Google Patents
Drug combination for eliminating neisseria gonorrhoeae resistance plasmids and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a drug combination for eliminating neisseria gonorrhoeae resistance plasmids and a preparation method thereof. The drug combination is composed of the following materials in parts by mass: 3-5 parts of golden cypress, 2-4 parts of rhizoma smilacis glabrae and 2-4 parts of astragalus root. The extract of the drug combination is prepared by a decoction method. The drug combination can effectively eliminate the neisseria gonorrhoeae resistance plasmids and recover sensitivity of gonococcus on antibiotics and particularly on cheap antibiotics (such as penicillin) to a certain extent, thus being of great significance to clinical application.
Description
Technical field
The present invention relates to medical technical field, be specifically related to a kind of pharmaceutical composition that can eliminate the Diplococcus gonorrhoeae R-plasmid and preparation method thereof.
Background technology
Gonorrhea is by Diplococcus gonorrhoeae (neisseria gonorrhoeae, be called for short NG) due to, although the sickness rate that since two thousand national most of areas should disease has downward trend, but according to " 2009 Chinese health statistics yearbook " data show, gonorrhea still is in higher morbidity level, is only second to syphilis in the sexually transmitted disease (STD).This disease is short incubation period, and infectiousness is strong, can cause many complication and sequela, and can concurrently infect other sexually transmitted disease (STD)s (comprising HIV) with promoting, consequence is serious.In recent years, because unreasonable application antibiotic, Diplococcus gonorrhoeae to the transformation and the chemical sproof increase of antibiotics sensitivity, cause the treatment of gonorrhea to select officina's limit, expense to rise, increased patient's financial burden.
Previously research prompting, the drug resistance of Diplococcus gonorrhoeae mainly is by plasmid-mediated, and plasmid-mediated drug resistance is the height drug resistance, and reason is the drug resistant gene that carries on the R-plasmid, given the drug resistance of bacterial antibiotic, plasmid is eliminated drug-resistance of bacteria and just can be lost thereupon.Therefore, stop the transmission of R-plasmid, recover it, have crucial meaning for the propagation for the treatment of and control gonorrhea to the antibiotic sensitivity of cheapness.Have the scholar to propose, Chinese medicine has certain elimination effect to the R-plasmid of some antibacterial (escherichia coli, dysentery bacterium, Pseudomonas aeruginosa, bacillus pyocyaneus, shigella etc.).The Chinese medicine of matter research of being used at present to disappear is of a great variety, as Rhizoma Atractylodis, Flos Lonicerae, Radix Scutellariae, Rhizoma Coptidis, Cortex Phellodendri, Herba Patriniae, compound recipe Pollen Typhae, Herba Houttuyniae, Radix Arnebiae (Radix Lithospermi) etc., but does not still have the report that Chinese medicine compound is studied Diplococcus gonorrhoeae R-plasmid elimination effect.
Summary of the invention
Problem to be solved by this invention is: how a kind of pharmaceutical composition of eliminating the Diplococcus gonorrhoeae R-plasmid and preparation method thereof that is used to is provided, this pharmaceutical composition can effectively be eliminated the Diplococcus gonorrhoeae R-plasmid, and can recover gonococcus to a certain extent to antibiotic sensitivity, the sensitivity of particularly cheap antibiotic (as penicillin), this is significant in clinical practice.
To achieve the above object of the invention, the technical solution adopted in the present invention is: a kind of pharmaceutical composition that is used to eliminate the Diplococcus gonorrhoeae R-plasmid is provided, it is characterized in that: each component that described pharmaceutical composition comprised and mass parts thereof are such as shown in down: 3～5 parts of Cortex Phellodendris; 2～4 parts of Rhizoma Smilacis Glabraes; 2～4 parts of the Radixs Astragali.
According to each component that the pharmaceutical composition preferred version comprised that is used to eliminate the Diplococcus gonorrhoeae R-plasmid provided by the present invention and mass parts thereof such as shown in down: 4 parts of Cortex Phellodendris; 3 parts of Rhizoma Smilacis Glabraes; 3 parts of the Radixs Astragali.
The above-mentioned preparation of drug combination method that is used to eliminate the Diplococcus gonorrhoeae R-plasmid, it is characterized in that: described preparation method comprises the steps:
A, take by weighing medical material with every part of 10g, and pulverizing medicinal materials is become the big fractionlet of the grain of rice according to prescription, standby;
The water of B, 3～5 times of amounts of adding decocted 20～40 minutes, afterwards filtering residue was separated with filtrate;
Add the water of 2～4 times of amounts in C, the filtering residue, decocted once more 20～40 minutes, afterwards filtering residue is separated with filtrate gained among the step B;
Add the water of 1～3 times of amount in D, the filtering residue, decocted once more 10～20 minutes, afterwards filtering residue is separated with filtrate gained among the step C;
E, the filtrate of gained among step B, C and the D is merged, vacuum filtration, constant temperature is concentrated into 33ml under 100 ℃ temperature;
F, the concentrated medicament of step e gained carried out packing after, behind the flowing steam sterilization 1h, cold preservation gets final product under 5 ℃ of temperature.
Pharmaceutical composition that is used to eliminate the Diplococcus gonorrhoeae R-plasmid provided by the present invention and preparation method thereof has following advantage:
1, can effectively eliminate the Diplococcus gonorrhoeae R-plasmid;
2, can recover gonococcus to a certain extent to antibiotic sensitivity, the sensitivity of particularly cheap antibiotic (as penicillin), this is significant in clinical practice.
Description of drawings
Fig. 1 is the preparation of drug combination flow chart that is used to eliminate the Diplococcus gonorrhoeae R-plasmid provided by the present invention.
The specific embodiment
Below in conjunction with accompanying drawing the present invention is further described:
Each component that pharmaceutical composition comprised and the mass parts ratio thereof that is used to eliminate the Diplococcus gonorrhoeae R-plasmid provided by the present invention is: 3～5 parts of Cortex Phellodendris; 2～4 parts of Rhizoma Smilacis Glabraes; 2～4 parts of the Radixs Astragali.
Above-mentionedly be used to eliminate each component that pharmaceutical composition comprised of Diplococcus gonorrhoeae R-plasmid and the preferred version of mass parts ratio is: 4 parts of Cortex Phellodendris; 3 parts of Rhizoma Smilacis Glabraes; 3 parts of the Radixs Astragali.
As shown in Figure 1, the above-mentioned preparation of drug combination method that is used to eliminate the Diplococcus gonorrhoeae R-plasmid comprises the steps: A, takes by weighing medical material according to prescription with every part of 10g, and pulverizing medicinal materials is become the big fractionlet of the grain of rice, and is standby; The water of B, 3～5 times of amounts of adding decocted 20～40 minutes, afterwards filtering residue was separated with filtrate; Add the water of 2～4 times of amounts in C, the filtering residue, decocted once more 20～40 minutes, afterwards filtering residue is separated with filtrate gained among the step B; Add the water of 1～3 times of amount in D, the filtering residue, decocted once more 10～20 minutes, afterwards filtering residue is separated with filtrate gained among the step C; E, the filtrate of gained among step B, C and the D is merged, vacuum filtration, constant temperature is concentrated into 33ml under 100 ℃ temperature; F, the concentrated medicament of step e gained carried out packing after, behind the flowing steam sterilization 1h, cold preservation gets final product under 5 ℃ of temperature.
Embodiment 1:
Prescription: Cortex Phellodendri 30g; Rhizoma Smilacis Glabrae 20g; Radix Astragali 20g.
Preparation method: A, take by weighing medical material with every part of 10g, and pulverizing medicinal materials is become the big fractionlet of the grain of rice according to prescription, standby; The water of B, 3 times of amounts of adding decocted 20 minutes, afterwards filtering residue was separated with filtrate; C, with adding the water of 2 times of amounts in the filtering residue of gained among the step B, decocted once more 20 minutes, afterwards filtering residue is separated with filtrate; D, with adding the water of 1 times of amount in the filtering residue of gained among the step C, decocted once more 10 minutes, afterwards filtering residue is separated with filtrate; E, the filtrate of gained among step B, C and the D is merged, vacuum filtration, constant temperature is concentrated into 33ml under 100 ℃ temperature; F, the concentrated medicament of step e gained carried out packing after, behind the flowing steam sterilization 1h, cold preservation gets final product under 5 ℃ of temperature.
Embodiment 2:
Prescription: Cortex Phellodendri 50g; Rhizoma Smilacis Glabrae 40g; Radix Astragali 40g.
Preparation method: A, take by weighing medical material with every part of 10g, and pulverizing medicinal materials is become the big fractionlet of the grain of rice according to prescription, standby; The water of B, 5 times of amounts of adding decocted 40 minutes, afterwards filtering residue was separated with filtrate; C, with adding the water of 4 times of amounts in the filtering residue of gained among the step B, decocted once more 40 minutes, afterwards filtering residue is separated with filtrate; D, with adding the water of 3 times of amounts in the filtering residue of gained among the step C, decocted once more 20 minutes, afterwards filtering residue is separated with filtrate; E, the filtrate of gained among step B, C and the D is merged, vacuum filtration, constant temperature is concentrated into 33ml under 100 ℃ temperature; F, the concentrated medicament of step e gained carried out packing after, behind the flowing steam sterilization 1h, cold preservation gets final product under 5 ℃ of temperature.
Embodiment 3:
Prescription: Cortex Phellodendri 40g; Rhizoma Smilacis Glabrae 30g; Radix Astragali 30g.
Preparation method: A, take by weighing medical material with every part of 10g, and pulverizing medicinal materials is become the big fractionlet of the grain of rice according to prescription, standby; The water of B, 4 times of amounts of adding decocted 30 minutes, afterwards filtering residue was separated with filtrate; C, with adding the water of 3 times of amounts in the filtering residue of gained among the step B, decocted once more 30 minutes, afterwards filtering residue is separated with filtrate; D, with adding the water of 2 times of amounts in the filtering residue of gained among the step C, decocted once more 15 minutes, afterwards filtering residue is separated with filtrate; E, the filtrate of gained among step B, C and the D is merged, vacuum filtration, constant temperature is concentrated into 33ml under 100 ℃ temperature; F, the concentrated medicament of step e gained carried out packing after, behind the flowing steam sterilization 1h, cold preservation gets final product under 5 ℃ of temperature.
To adopt disappear the respectively situation of matter test of pharmaceutical composition provided by the present invention and control drug to be described below below:
30 strains of isolation identification in outpatient service gonorrhea patient of adopting the dermatopathy and venereal disease study on prevention STD of institute of People's Hospital, Sichuan Prov. of Sichuan Academy of Medical Sciences laboratory to collect are produced the Diplococcus gonorrhoeae bacterial strain (PPNG) of beta-lactamase as experimental strain.The WHO that employing is provided by national sexually transmitted disease (STD) center recommends Diplococcus gonorrhoeae reference culture A, B, C, D, E as the Quality Control bacterial strain.Drug extract provided by the present invention and sodium lauryl sulphate (SDS) are adopted in the matter that disappears agent.
The matter that disappears experiment
The bacterial strain that is positive with the beta-lactam enzyme test is as the experiment bacterial strain.Observe the drug extract provided by the present invention matter effect that disappears: drug extract provided by the present invention is as experimental group, and the routine matter agent SDS that disappears organizes the matter experiment that disappears, each 30 strain of experimental strain, observed result behind the matter that the disappears 72h in contrast.
Adopt agar dilution to carry out drug sensitive experiment, measure Diplococcus gonorrhoeae penicillin, tetracycline, ciprofloxacin, spectinomycin and 5 kinds of antibiotic drug resistance spectrums of ceftriaxone.Xi Tai district's Diplococcus gonorrhoeae drug sensitivity tests criterion (being provided by national sexually transmitted disease (STD) center) is provided the drug resistance criterion.
It is 10 that Diplococcus gonorrhoeae bacterial strain after the recovery transferred species is prepared into concentration
5The Diplococcus gonorrhoeae liquid of/ml and be inoculated in the T-M culture medium 24h of drug extract provided by the present invention and SDS after observe the bacterial growth situation, be subclinical Mlc with antibacterial maximum growth concentration.
Plasmid table is measured before and after the matter that disappears: adopt " molecular cloning experiment guide " plasmid post test kit extracting Diplococcus gonorrhoeae plasmid before and after the matter that disappears, and analyze its molecular weight with agarose gel electrophoresis.
Experimental result
The matter that disappears agent subinhibitory concentration: the subclinical Mlc of drug extract provided by the present invention is 11.836mg/ml, and the subclinical Mlc of SDS is 0.15mg/ml.
Experimental strain is composed 5 kinds of common antibiotics drug resistances: the preceding 30 strain experimental strains of the matter that disappears test are all to spectinomycin and ceftriaxone sensitivity, to penicillin, tetracycline and ciprofloxacin drug resistance; The matter that disappears test back 30 strain experimental strains are still all to spectinomycin and ceftriaxone sensitivity, but the sensitivity of penicillin, tetracycline and ciprofloxacin is had in various degree recovery, specifically describe as table 1:
Table 1
The drug resistance of experimental strain relatively before and after the matter that disappears: adopt four fold table χ
2Assay shows, and compares before the matter that disappears, and the experimental strain after the drug extract effect provided by the present invention all descends to the resistant rate of penicillin, tetracycline and ciprofloxacin, and difference has statistical significance (the P value is all less than 0.01).Experimental strain after the SDS effect all descends to the resistant rate of penicillin and ciprofloxacin, difference has statistical significance (definite probabilistic method calculate to such an extent that P value is respectively 0.024 and 0.011), but can not think that still experimental strain after the SDS effect is to the resistant rate decline (P=0.492) of tetracycline.
The drug resistance of drug extract group provided by the present invention and SDS group experimental strain relatively after the matter that disappears: adopt four fold table χ
2Assay shows, experimental strain after drug extract provided by the present invention and the SDS effect is to the equal indifference of the drug resistance of penicillin and ciprofloxacin (the P value is respectively 0.152 and 0.165), and the experimental strain after the drug extract effect provided by the present invention is lower than (P=0.020) after the SDS effect to the resistant rate of tetracycline.Generally speaking, drug extract recovery gonococcus provided by the present invention is good than SDS to the effect of drug resistance antibiotics sensitivity.
The drug extract provided by the present invention matter effect that disappears:
Experimental strain plasmid table before the matter that disappears: the preceding 30 strain PPNG bacterial strains of the matter that disappears experiment all can detect 2～3 plasmid bands, and detecting plasmid has 4.2kb, 5.3kb, 7.4kb, and recall rate is respectively 100%, 63.3% and 86.7%.Plasmid table has three kinds, based on 4.2+5.3+7.4 and 4.2+7.4 (accounting for 86.7%).The plasmid table type of 30 strain PPNG bacterial strains sees Table 2.
Table 2
The matter that disappears effect: after drug extract provided by the present invention and SDS effect, have the plasmid band of 5 strains and 7 strain experimental strains losing in various degree to occur respectively.Table 3 for the matter agent effect that disappears after PPNG bacterial strain plasmid eliminate general status, table 4 be that drug extract provided by the present invention and SDS are to 4.2kb, 5.3kb, three kinds of plasmids eliminations of 7.4kb situation.
Table 3
Adopt χ
2Check, the result shows two groups of plasmid elimination factor difference not statistically significant (χ
2=0.417, P=0.519).
Table 4
Adopt χ
2Check, drug extract provided by the present invention and SDS are to the equal not statistically significant of elimination factor difference (the P value is all greater than 0.05) of 4.2kb, 5.3kb, 7.4kb, and it is different to think that still drug extract provided by the present invention and SDS eliminate the plasmid effect.
The PPNG that this research is adopted is to penicillin, tetracycline, the whole drug resistances of ciprofloxacin, but disappears after the matter effect through drug extract provided by the present invention, and resistant rate drops to 60～70% by original 100%, and difference has statistical significance.After the SDS effect, PPNG also drops to about 80% the resistant rate of penicillin and ciprofloxacin, and difference has statistical significance, but whether SDS can reduce the further research of still needing of the resistant rate of tetracycline.Result of study shows that Chinese medicine drug extract provided by the present invention can recover gonococcus to a certain extent to antibiotic sensitivity, the sensitivity of particularly cheap antibiotic (as penicillin), and this is significant in clinical practice.
Plasmid 4.2kb is a cryptic plasmid, and this plasmid is present in 96% the clinical Diplococcus gonorrhoeae separated strain, and other antibacterial does not contain this plasmid, and this experiment recall rate is 100%; 5.3kb, the 7.4kb plasmid is the penicillin resistant plasmid, the former detects in African country, be by " African type ", the latter is " Asian type ", the penicillin drug resistance that these two kinds of plasmids can be encoded plasmid-mediated, the recall rate of these two kinds of plasmids is respectively 63.3% and 86.7% in this experiment, and this conforms to the penicillin drug resistance with Diplococcus gonorrhoeae.After the matter that the disappears effect, 4.2kb, three kinds of plasmids of 5.3kb, 7.4kb all have elimination in various degree, drug extract wherein provided by the present invention reaches about 20% the elimination factor of 5.3kb and 7.4kb, and this may recover relevant to the mechanism of penicillin sensitivity with Diplococcus gonorrhoeae.Disappeared the matter effect after 72 hours through drug extract provided by the present invention and SDS, and external plasmid elimination factor is respectively 16.7% and 23.3%, and the difference not statistically significant can think that the effect of elimination plasmid of drug extract provided by the present invention and SDS is suitable.At present result of study shows that the plasmid elimination factor has than great fluctuation process, and different Chinese medicines are to the R-plasmid elimination factor difference of same antibacterial, and also there is bigger difference to same medicine in different researcher to the result of study of same antibacterial.
Because SDS is harmful chemical reagent, does not have clinical practice, by contrast, drug extract provided by the present invention can be for human body use and cheap, safer, economic.
Claims (3)
1. pharmaceutical composition that is used to eliminate the Diplococcus gonorrhoeae R-plasmid is characterized in that: each component that described pharmaceutical composition comprised and mass parts thereof are such as shown in down:
3～5 parts of Cortex Phellodendris;
2～4 parts of Rhizoma Smilacis Glabraes;
2～4 parts of the Radixs Astragali.
2. according to the described pharmaceutical composition that is used to eliminate the Diplococcus gonorrhoeae R-plasmid of claim 1, it is characterized in that each component that described pharmaceutical composition comprised and mass parts thereof are such as shown in down:
4 parts of Cortex Phellodendris;
3 parts of Rhizoma Smilacis Glabraes;
3 parts of the Radixs Astragali.
3. claim 1 or the 2 described preparation of drug combination methods that are used to eliminate the Diplococcus gonorrhoeae R-plasmid, it is characterized in that: described preparation method comprises the steps:
A, take by weighing medical material with every part of 10g, and pulverizing medicinal materials is become the big fractionlet of the grain of rice according to prescription, standby;
The water of B, 3～5 times of amounts of adding decocted 20～40 minutes, afterwards filtering residue was separated with filtrate;
Add the water of 2～4 times of amounts in C, the filtering residue, decocted once more 20～40 minutes, afterwards filtering residue is separated with filtrate gained among the step B;
Add the water of 1～3 times of amount in D, the filtering residue, decocted once more 10～20 minutes, afterwards filtering residue is separated with filtrate gained among the step C;
E, the filtrate of gained among step B, C and the D is merged, vacuum filtration, constant temperature is concentrated into 33ml under 100 ℃ temperature;
F, the concentrated medicament of step e gained carried out packing after, behind the flowing steam sterilization 1h, cold preservation gets final product under 5 ℃ of temperature.
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| 陆原，等.尿路清抗淋球菌作用的体外试验.《中国麻风皮肤病杂志》.2005,第21卷(第10期),796-797. * |
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