CN109568494A - A kind of application of bletilla alcohol extract in preparation polymyxins Trimethoprim - Google Patents
A kind of application of bletilla alcohol extract in preparation polymyxins Trimethoprim Download PDFInfo
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Abstract
The invention discloses a kind of application of bletilla alcohol extract in preparation polymyxins Trimethoprim, and the synergist is to mix bletilla alcohol extract with polymyxin B, and wherein bletilla alcohol extract and polymyxin B mass ratio are 1~256: 1.Bletilla alcohol extract provided by the invention be used alone it is weak to the inhibiting effect of Escherichia coli, but with antibacterial activity that the latter can be remarkably reinforced after polymyxin B use in conjunction, can especially enhance polymyxin B to 2-4 times of antibacterial activity of drug resistance Escherichia coli.
Description
(1) technical field
The present invention relates to a kind of bletilla alcohol extract, in particular to a kind of bletilla alcohol extract is in preparation polymyxins antibacterial synergy
Application in agent.
(2) background technique
Escherichia coli (Escherichia coli) is commonly called as Escherichia coli, is the normal bacterium in humans and animals enteron aisle
Group.But the Escherichia coli of some serotypes can often cause various enteron aisles and parenterally disease, such as diarrhea, hemorrhagic intestines
Inflammation, neonatal purulent meningitis, urinary tract infections or even septicemia etc..With severe sense caused by mechanism in multiple antibiotic resistant Escherichia coli
It contaminates increasing, while lacking again and can fight the new antibiotic of this infection, currently, polymyxins, which has become, treats large intestine bar
The last line of defense of bacterium.Although it is lower drug resistant probability occur according to previous report polymyxins, and is all Bacterial stain
Gene mutation causes, will not propagate between different bacterium on body.However, what is at home and abroad found recently is isolated from animal and patient
Escherichia coli in detect plasmid-mediated super drug resistant gene mcr-1, not only make Escherichia coli generate polymyxins resistance,
And it can the horizontal transmission in different strains.To make clinical treatment coli-infection become extremely complex and difficult, even
It is on the verge of the available condition of no medicine.Therefore, new strategy and method is explored to have become a top priority.
Bletilla is a precious Plant's kind during orchid bletilla belongs to, and has long usage history, first recorded in " mind
Agriculture book on Chinese herbal medicine warp ", it is recorded in the Chinese medical books such as " book on Chinese herbal medicine readability ", Compendium of Material Medica thereafter.Bletilla is distributed in lung channel, cures mainly and coughs up
The diseases such as blood, Carbuncle swells dislikes sore；It also is the main ingredient of ancient Chinese medicine doctor treatment tuberculosis (pulmonary tuberculosis).Modern pharmacological studies have shown that bletilla is rich in
Antibacterial Constituents, such as luxuriant and rich with fragrance class and Bibenzyl compound.The present invention is using Pseudobulbus Bletillae (Rhizoma Bletillae) extract as the anti-Escherichia coli of polymyxins
A kind of synergist, lay the foundation for the research and development of novel antibacterial Chinese medicine.
(3) summary of the invention
The invention aims to solve the problems, such as the Drug Resistance of E. coli got worse, and provide a kind of bletilla alcohol extract
And its application of the Trimethoprim as polymyxins.
The technical solution adopted by the present invention is that:
The present invention provides a kind of application of the bletilla alcohol extract in preparation polymyxins Trimethoprim, the synergy
Agent is to mix bletilla alcohol extract with polymyxin B, and wherein bletilla alcohol extract and polymyxin B mass ratio are 1~256:
1.The synergist refers to Escherichia coli sensitive to polymyxins and the antibacterial synergy to the drug resistant Escherichia coli of polymyxins
Agent, wherein it is to carry plasmid-mediated super drug resistant gene mcr-1 that more plants, which are clinically separated bacterium,.
Bletilla alcohol extract concentration is 1-256 μ g/mL in synergist of the present invention, and polymyxin B concentration is 0.5-4 μ g/
ML, e. coli concentration 106cfu/ml。
Bletilla alcohol extract of the present invention is prepared as follows: after bletilla drying and crushing, with 95% second of volumetric concentration
(being preferably concentrated into 1/3~1/20 times of volume) is concentrated in alcohol solution refluxing extraction, filtrate decompression, using polyamide column chromatography, first
It is washed with deionized water and takes off to after colourless, then eluted with the ethanol water of volumetric concentration 20-60%, collect target components eluting peak
(eluent of 60% ethanol water of preferred volume concentration), is concentrated to dryness, and obtains bletilla alcohol extract.
Further, the bletilla drying and crushing, which refers to, dries bletilla root or dries in 30-80 DEG C of baking oven, crushes, mistake
10-100 mesh.
Further, the ethanol water volumetric usage is calculated as 3-10ml/g with bletilla powder weight.
Further, refluxing extraction 3 times preferably described, each 30-120min.
Further, the polyamide column chromatography method are as follows: polyamide is added (preferably in the concentrate after filtrate is concentrated
200-300 mesh) it mixes thoroughly, it is reloaded into progress column chromatography in the chromatographic column added with polyamide.
Further, it is preferably eluted with the ethanol water of volumetric concentration 60%, collects 60% ethanol elution peak, be concentrated under reduced pressure
To dry, acquisition bletilla alcohol extract.
Compared with prior art, beneficial effect of the present invention is mainly reflected in: bletilla alcohol extract provided by the invention individually makes
It is weak with the inhibiting effect to Escherichia coli, but with can be remarkably reinforced after polymyxin B use in conjunction the latter antibacterial activity (see
Table 1), it can especially enhance polymyxin B to 2-4 times of antibacterial activity of drug resistance Escherichia coli.
(4) Detailed description of the invention
Fig. 1 bletilla alcohol extract (EF60) HPLC analyzes map.
Fig. 2 bletilla alcohol extract (EF60) and polymyxins are combined to resistance to polymyxins Escherichia coli MCR1_NJ (A) and large intestine
The influence of bacillus ATCC 25922 (B) growth；
The time-kill curve of Fig. 3 Escherichia coli MCR1_NJ (A) and Escherichia coli ATCC25922 (B).
(5) specific embodiment
The present invention is described further combined with specific embodiments below, but protection scope of the present invention is not limited in
This:
Bletilla of the present invention is by " Chinese Pharmacopoeia " 2015 editions acquisition preparations.
The preparation of 1 bletilla alcohol extract of embodiment
Bletilla is dried in 60 DEG C of baking ovens, is crushed, 40 meshes is crossed, 1kg dried powder is taken to add body by material ratio 1kg:4L
Product 95% ethanol water refluxing extraction of concentration 3 times, each 90min.Merging filtrate is concentrated under reduced pressure, and obtains 110g alcohol extract, adds
Enter 100g polyamide (200-300 mesh) to mix thoroughly, go on the chromatographic column (10cm X 40cm) for having been loaded with 600g polyamide, to go
Ionized water be eluted to it is colourless after, first with 20% ethanol water of volumetric concentration rinse 5 column volumes, then use volumetric concentration 60%
Ethanol water elute 3 column volumes, collect 60% ethanol elution peak, be concentrated to dryness and (be denoted as to get bletilla alcohol extract
EF60) 15g, the position are rich in luxuriant and rich with fragrance class compound, and HPLC map is shown in Fig. 1.
2 Antibiotics resistance test of embodiment
This experimental example is to study bletilla alcohol extract EF60 from polymyxins to different Escherichia coli minimum inhibitory concentrations.
It is inoculated in MH plate respectively for examination bacterium by various in table 1, places in 37 DEG C of incubators and cultivates 48h, chosen from plate
It selects on the single colonie access inclined-plane MH, 37 DEG C of cultures are for 24 hours, spare.
From a ring is chosen on the inclined-plane of activation for trying bacterium into 5ml, MH fluid nutrient medium, 37 DEG C, 160rpm shaken cultivation mistake
At night, being diluted to bacterial concentration with MH fluid nutrient medium is 106Cfu/ml, as trying bacterium solution.Most using the measurement of two-fold dilution's method
Small Mlc, operation all carry out in super-clean bench.Untested compound is diluted to different final concentration (64 μ with MH culture medium
G/mL EF60,1 μ g/mL polymyxin B, 64 μ g/mL EF60+1 μ g/mL polymyxin Bs), respectively take 100 μ L that 96 orifice plates are added
In, 100 μ L are then added in every hole for trying bacterium solution, mixing, which is placed in 37 DEG C of incubator, cultivates 20 hours, surveys OD600Value, together
Shi Caiyong is visually observed, and is set to minimum inhibitory concentration to completely inhibit the minimum concentration of indicator bacteria growth, is recorded EF60 and glue more
MIC when rhzomorph B is individually and two medicines are combined.
MH plate quality composition: 0.2% powdered beef, 0.15% soluble starch, 1.75% acid hydrolyzed casein, 1.0%
Agar, solvent are deionized water, and pH value is natural.
MH fluid nutrient medium quality composition: 0.2% powdered beef, 0.15% soluble starch, 1.75% acid hydrolyzed casein,
Solvent is deionized water, and pH value is natural.
The results are shown in Table 1, when exclusive use, MIC >=256 μ g/ of the bletilla alcohol extract EF60 to all Escherichia coli
ML, antibacterial action are weaker；Polymyxin B is sensitive (MIC=1) to ATCC 25922 and zd01 strains expressed, but to other bacterium
Strain shows as drug resistance (MIC=4 μ g/mL).When the two combination, two plants of bacterium is not only made at least to improve 4 to the sensibility of EF60
Times, and more plants of resistance to polymyxins Escherichia coli is made to drop to 1 from 4 to the MIC of polymyxin B, i.e., it is changed into sensitivity from drug resistance.
1 EF60 of table and polymyxin B are applied alone and are combined MIC and FICI
3 growth inhibition curve of experimental example
This experimental example is the polymyxins drug combination for studying bletilla alcohol extract EF60 and subinhibitory concentration to large intestine bar
The inhibitory effect of bacterium growth.
Escherichia coli MCR1_NJ is inoculated in MH plate, places in 37 DEG C of incubators and cultivates 48h, one is selected from plate
On a single colonie access inclined-plane MH, 37 DEG C of cultures are for 24 hours, spare.A ring is chosen from the inclined-plane of activation for examination bacterium to 5ml, MH liquid
In culture medium, 37 DEG C, 160rpm shaken cultivation is stayed overnight, and being diluted to bacterial concentration with MH culture medium is 106Cfu/ml obtains large intestine
Bacillus MCR1_NJ bacterium solution.
EF60 and polymyxin B prepared by embodiment 1 are diluted to various concentration (the 1 more Acarasiales of μ g/mL with MH culture medium
Plain B, 0.5 μ g/mL polymyxin B ,+64 μ g/mL EF60 of 1 μ g/mL polymyxin B ,+64 μ g/mL of 0.5 μ g/mL polymyxin B
EF60,64 μ g/mL EF60), it respectively takes 100 μ L to be added in 96 orifice plates, 100 μ L Escherichia coli MCR1_NJ is then added in every hole
Bacterium solution, mixing, which is placed in 37 DEG C of incubator, cultivates 20 hours, surveys OD600Value, while with the MH of the 5%DMSO containing volumetric concentration training
It supports base to compare as not dosing, as a result as shown in A in Fig. 2.
Escherichia coli MCR1_NJ is replaced to prepare 10 with Escherichia coli ATCC25922 under similarity condition6Cfu/ml Escherichia coli
EF60 and polymyxin B prepared by embodiment 1 are diluted to various concentration (0.5 μ g/mL with MH culture medium by ATCC25922 bacterium solution
Polymyxin B, 0.25 μ g/mL polymyxin B ,+16 μ g/mL EF60 of 0.5 μ g/mL polymyxin B, the 0.25 more Acarasiales of μ g/mL
Plain B+16 μ g/mL EF60,16 μ g/mL EF60), it respectively takes 100 μ L to be added in 96 orifice plates, it is big that 100 μ L is then added in every hole
Enterobacteria ATCC25922 bacterium solution, mixing, which is placed in 37 DEG C of incubator, cultivates 20 hours, surveys OD600Value, while to contain 5%
The MH culture medium of DMSO is compareed as not dosing, as a result as shown in B in Fig. 2.
The basic unrestraint of Escherichia coli MCR1_NJ is made as shown in Fig. 2, 64 μ g/mL (1/4 × MIC) EF60 are used alone
With.Have more significantly in 0-4h to the growth of MCR1_NJ when being combined with 0.5 μ g/mL (1/8 × MIC) polymyxin B
Inhibiting effect；And the growth of the antibody-resistant bacterium is then completely inhibited when being combined with 1 μ g/mL (1/4MIC) polymyxin B.It is similar
, to the early stage of Escherichia coli ATCC 25922 when 16 μ g/mL EF60 and 0.25 μ g/mL (1/4 × MIC) polymyxin B are combined
Growth has certain inhibiting effect, but over time, thallus rapid growth again；When polymyxin B is increased to 0.5 μ g/mL
When (1/2 × MIC), the two combination could completely inhibit 25922 strain growth of Escherichia coli ATCC.The above results are further true
Demonstrate,proving EF60 and polymyxin B combination has collaboration bacteriostasis to Escherichia coli MCR1_NJ, and to Escherichia coli ATCC25922
Only there is addition antibacterial action.
4 time-kill curve of experimental example
This experimental example is to study bletilla alcohol extract EF60 and polymyxins drug combination to Escherichia coli bactericidal effect.
Escherichia coli MCR1_NJ is inoculated in MH plate, places in 37 DEG C of incubators and cultivates 48h, one is selected from plate
On a single colonie access inclined-plane MH, 37 DEG C of cultures are for 24 hours, spare.A ring is chosen from the inclined-plane of activation for examination bacterium to 5ml, MH liquid
In culture medium, 37 DEG C, 160rpm shaken cultivation is stayed overnight, and being diluted to bacterial concentration with culture medium is about 106Cfu/ml obtains large intestine
Bacillus MCR1_NJ bacterium solution.
EF60 and polymyxin B prepared by embodiment 1 are diluted to various concentration (the 1 more Acarasiales of μ g/mL with MH culture medium
Plain B, 4 μ g/mL polymyxin Bs ,+64 μ g/mL EF60 of 1 μ g/mL polymyxin B ,+64 μ g/mL of 4 μ g/mL polymyxin B
EF60,64 μ g/mL EF60), it respectively takes 100 μ L to be added in 96 orifice plates, 100 μ L Escherichia coli MCR1_NJ is then added in every hole
Bacterium solution, mixing, which is placed in 37 DEG C of incubator, cultivates 20 hours, surveys OD600Value, at the same using the MH culture medium containing 5%DMSO as
Not dosing control, as a result as shown in A in Fig. 3.
Escherichia coli MCR1_NJ is replaced to prepare 10 with Escherichia coli ATCC25922 under similarity condition6Cfu/ml Escherichia coli
EF60 and polymyxin B prepared by embodiment 1 are diluted to various concentration (0.5 μ g/mL with MH culture medium by ATCC25922 bacterium solution
Polymyxin B, 1 μ g/mL polymyxin B ,+16 μ g/mL EF60 of 0.5 μ g/mL polymyxin B ,+16 μ of 1 μ g/mL polymyxin B
G/mL EF60,16 μ g/mL EF60), it respectively takes 100 μ L to be added in 96 orifice plates, 100 μ L Escherichia coli is then added in every hole
ATCC25922 bacterium solution, mixing, which is placed in 37 DEG C of incubator, cultivates 20 hours, surveys OD600Value, while with the MH training containing 5%DMSO
It supports base to compare as not dosing, as a result as shown in B in Fig. 3.
As shown in figure 3, when 64 μ g/mL EF60 and 1 μ g/mL polymyxin B are used alone, to Escherichia coli MCR1_NJ
Equal unrestraint effect can completely inhibit bacterium growth, but without bactericidal effect when the two combination；As 64 μ g/mLEF60 and 4 μ g/
When mL (1 × MIC) polymyxin B is combined, then it can be seen that EF60 can enhance sterilization of the polymyxin B to Escherichia coli MCR1_NJ
Speed.The bactericidal effect of this enhancing polymyxin B embodies more abundant on Escherichia coli ATCC 25922.Such as 1 μ g/mL
When (1 × MIC) polymyxin B is used alone, to ATCC 25922 substantially without any bactericidal effect；But with 16 μ g/mL EF60
When combination, ATCC 25922 is just nearly no detectable surviving colonies number after 3h is handled.
5 Mouse Protection Test of experimental example
ICR mouse 70 are taken, male, weight 18-22g is randomly divided into control group, Escherichia coli group, bletilla alcohol extract
EF60 group, polymyxin B group, bletilla alcohol extract EF60+ polymyxin B group.Every group of 10 animals.Any examination is not given in control
Agent；Escherichia coli group, intraperitoneal injection 2 × 107/ kg Escherichia coli MCR1_NJ is (dilute with 1% yeast physiological saline suspension (w/v)
It releases)；Bletilla alcohol extract EF60 group, intraperitoneal injection 2 × 107After Escherichia coli MCR1_NJ half an hour/kg living, intraperitoneal injection
60mg/kg bletilla alcohol extract (is prepared) with 0.2% sodium carboxymethylcellulose and 0.01% Tween 80；Polymyxin B group, abdominal cavity note
Penetrate 2 × 107After Escherichia coli MCR1_NJ half an hour/kg living, tail vein injection 0.5mg/kg polymyxin B (physiological saline
It prepares)；Bletilla alcohol extract EF60+ polymyxin B group, gives 2 × 10 respectively7Escherichia coli MCR1_NJ half an hour/kg living
Afterwards, 10,30,60mg/kg bletilla alcohol extract EF60 are then injected intraperitoneally, even in difference tail vein injection 0.5mg/kg polymyxin B
Continuous administration 3 days, once a day.Normal diet and drinking-water, mouse ordinary circumstance and death in observation 14 days are given after administration
Rate (table 2).Bletilla alcohol extract joint polymyxin B can obviously reduce the death of infection antibiotic-resistance E. coli mouse as the result is shown
Rate shows that bletilla alcohol extract and polymyxin B combination have synergistic protective effect.
2 bletilla alcohol extract of table and polymyxins are combined the influence to coli-infection mouse death rate
Note: X2It examines, P < 0.001 * (compared with Escherichia coli group).
Claims (10)
1. a kind of application of bletilla alcohol extract in preparation polymyxins Trimethoprim.
2. application as described in claim 1, it is characterised in that the synergist mixed with polymyxin B by bletilla alcohol extract and
At wherein bletilla alcohol extract and polymyxin B mass ratio are 1~256: 1.
3. application as described in claim 1, it is characterised in that the synergist is Escherichia coli Trimethoprim, the large intestine
Bacillus is polymyxins responsive type or drug-resistant type.
4. application as claimed in claim 3, it is characterised in that the Escherichia coli carry super drug resistant gene mcr-1.
5. application as claimed in claim 2, it is characterised in that bletilla alcohol extract concentration is 1-256 μ g/mL in the synergist.
6. application as described in claim 1, it is characterised in that the bletilla alcohol extract is prepared as follows: bletilla is dry
After crushing, using polyamide column chromatography, first used after filtrate decompression concentration with 95% ethanol water refluxing extraction of volumetric concentration
Deionized water be eluted to it is colourless after, then eluted with the ethanol water of volumetric concentration 20-60%, collect target components eluting peak,
It is concentrated to dryness, obtains bletilla alcohol extract.
7. application as claimed in claim 6, it is characterised in that the bletilla drying and crushing, which refers to, dries bletilla root or in 30-
It dries, crushes in 80 DEG C of baking ovens, cross 10-100 mesh.
8. application as claimed in claim 6, it is characterised in that the ethanol water volumetric usage is with bletilla powder weight
For 3-10ml/g.
9. application as claimed in claim 6, it is characterised in that the refluxing extraction 3 times, each 30-120min.
10. application as claimed in claim 6, it is characterised in that the ethanol water volumetric concentration 60% of elution.
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