CN102911257A - Cyclic lipopeptide antibiotic and preparation and application thereof - Google Patents
Cyclic lipopeptide antibiotic and preparation and application thereof Download PDFInfo
- Publication number
- CN102911257A CN102911257A CN2012103567857A CN201210356785A CN102911257A CN 102911257 A CN102911257 A CN 102911257A CN 2012103567857 A CN2012103567857 A CN 2012103567857A CN 201210356785 A CN201210356785 A CN 201210356785A CN 102911257 A CN102911257 A CN 102911257A
- Authority
- CN
- China
- Prior art keywords
- cyclic lipopeptide
- preparation
- cyclic
- aqueous solution
- antibiotic
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Landscapes
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention relates to a novel cyclic lipopeptide antibiotic (cyclic nonapeptide avermectin) and preparation and application thereof, belonging to the technical field of biomedicine. The fermentation medium is inoculated with paenibacillus, the paenibacillus is cultured at 28-35 DEG C for 24-96 hours, the fermentation broth is centrifuged to take the supernatant, the supernatant is adsorbed by a macroporous adsorption resin, and separation and purification are performed further to obtain the cyclic lipopeptide antibiotic which can be used for preparing the bacterial-infection resisting medicament. The invention provides the cyclic nonapeptide avermectin with higher toxicity than polymyxin B and higher medical application value and the preparation and application of the cyclic nonapeptide avermectin. The experiments in vitro prove that the compound can inhibit all the tested Gram-positive and negative pathogens including the multiple and super medicament resisting bacteria significantly, and the in-vivo experiments prove that the compound can prevent and treat the mouse septicemia caused by theextended-spectrum medicament resisting pseudomonas aeruginosa significantly.
Description
Technical field
The present invention relates to a kind of new cyclic lipopeptide microbiotic-ring nonapeptide rhzomorph and preparation and application thereof, belong to field of biomedicine technology.
Background technology
Because antibiotic widespread use or abuse have occurred losing efficacy widely so that once play the microbiotic of remarkable effect in the bacterial infection disease treatment.Many common bacterias constantly strengthen the resistance of common antibiotics.At present, the bacterial drug resistance serious threat mankind's health.The methicillin-resistant staphylococcus aureus, the multi-drug resistant bacterias such as the escherichia coli of penicillin resistance pneumococcus and product extended spectrumβ-lactamase (ESBLs) and klebsiella have become spreading rate and the very high pathogenic agent of lethality rate in the hospital.The quick appearance of vancomycin-resistant enterococcus (VRE) has aggravated the worry of people to pathogenic bacterium resistance problem.
In addition, along with the widespread use of immunosuppressor and Broad spectrum antibiotics, the popularization of conduit, intubate, burn rescue, chemicotherapy and organ transplantation, the sickness rate of fungi infestation also increases year by year.According to incompletely statistics, over nearly 10 years, the sickness rate of systemic fungal infection has increased by 3～5 times, and poor prognosis, mortality ratio are high.According to chemical structure, the antifungal drug of listing can be divided into four large classes: propylene class (Terbinafine), polyenoid class (amphotericin B etc.), azole (itraconazole etc.), echinocandin class (Caspofungin) etc.The Terbinafine curative effect is low, can not be individually dosed; The polyenoid class has serious untoward reaction; Azole only can suppress and can not killing fungus; Although the echinocandin class is all effective for Candida and Eurotium, and security is higher, faces and there is no oral dosage form and the two large problems such as expensive, so limited their application clinically.
Given this, the new antibiotic that seek novel structure, is difficult for producing crossing drug resistant and suitable suitability for industrialized production will be the long-term and difficult task of of facing mankind.
Summary of the invention
The objective of the invention is to overcome above-mentioned weak point, provide a kind of for the clinical drug-resistant pathogenic bacterium, have the new lipopeptide antibiotic that potential medical use is worth---preparation method and the application of ring nonapeptide rhzomorph.
According to technical scheme provided by the invention, a kind of cyclic lipopeptide microbiotic, skeleton symbol is as follows:
Wherein L-Dab is the L-type 2,4-diamino-butanoic, and L-Ile is the L-type Isoleucine, and D-Phe is D type phenylalanine, and L-Leu is the L-type leucine, and D-Val is D type α-amino-isovaleric acid, and L-Thr is the L-type Threonine.
Described bacterial strain is series bacillus (Paenibacillus ehirnensis strain B7), be preserved in Chinese Typical Representative culture collection center, preservation date: on June 24th, 2012, the address: Wuhan, China Wuhan University, preserving number is CCTCC NO:M 2012244.
The antibiotic preparation method of cyclic lipopeptide, step is:
(1) cultivation and fermentation: series bacillus (Paenibacillus ehirnensis strain B7) is inoculated into fermention medium, obtain activating bacterial strain, activation bacterial strain twice of shaking culture in seed culture medium, obtain secondary seed solution, shaking culture fermentation in the secondary seed solution access fermention medium, obtain fermented liquid, centrifugal, get fermented liquid supernatant, go precipitation, through absorption with macroporous adsorbent resin, the fermentation supernatant is flowed through with distilled water balance chromatography column good, that be filled with macroporous adsorbent resin with appropriate speed first;
(2) separation and purification: after complete on the sample, respectively with distilled water and methanol aqueous solution flushing, then use the methanol aqueous solution wash-out first; Collect each elution peak, survey and live, the concentrated evaporate to dryness of active ingredient is obtained crude product; With the crude product dissolve with methanol, centrifuging is further purified with solid-phase extraction column; Carry out consummately with preparative high performance liquid chromatography again, collect active ingredient, concentrated, freeze-drying can obtain described ring nonapeptide rhzomorph;
The antibiotic preparation method of described cyclic lipopeptide, concrete steps are:
The series bacillus (Paenibacillus ehirnensis strain B7) that (1) will be stored in the glycerine pipe of-80 ℃ of refrigerators is transferred on the nutrient agar, cultivates 24～96h, obtains activating bacterial strain for 28～35 ℃;
Nutrient agar is prepared by following composition: extractum carnis 3g, peptone 10g, sodium-chlor 5g, agar 17g, distilled water 1000mL, pH7.2;
(2) will activate the bacterial strain access and be contained in the 50mL seed culture medium of 250mL triangular flask, in 28～35 ℃, 220rpm shaking culture 24h is primary seed solution;
(3) the primary seed solution access with step (2) gained is contained in the 200mL seed culture medium of 500mL triangular flask, and in 28～35 ℃, 220rpm shaking culture 24h is secondary seed solution;
Seed culture medium is nutrient broth medium, prepares by following composition: extractum carnis 3g, peptone 10g, sodium-chlor 5g, tap water 1000mL, pH7.2;
(4) by volume 5%～10% the inoculum size access of secondary seed solution with step (3) gained contains in the 2L triangular flask of 500mL fermention medium, and in 28～35 ℃, 200rpm shaking culture 96h obtains fermented liquid;
Fermention medium is prepared by following composition: glucose, 0～10g/L; CaCl
2, 0.01～0.7g/L; MgSO
47H
2O, 0.05～1g/L; ZnSO
4, 0.005～0.1g/L; NaCl, 0.05～0.5g/L; FeSO
4, 0.005～0.05g/L; (NH
4)
2SO
4, 0.5～4.0g/L; MnSO
4H
2O, 0.001～0.01g/L; KH
2PO
4, 0.5～5g/L; Solvent is distilled water, and pH 7.0～7.5;
(5) fermented liquid obtained of step (4) forwards in the centrifugal bottle, in the centrifugal 30min of 6500 * g, removes precipitation after the balance, gets the clarification fermented supernatant fluid;
(6) supernatant liquor of step (5) gained is worn the good Amberlite XAD-16 chromatography column of distilled water balance with the flow velocity stream of 10～50mL/min, complete rear respectively with distilled water and to contain volume ratio be that the aqueous solution of 45% methyl alcohol respectively washes 5 bed volumes on sample, then with containing the aqueous solution wash-out that volume ratio is 90% methyl alcohol, collected volume is than the elution peak that is 90% methanol aqueous solution wash-out, 55～65 ℃ of concentrated evaporates to dryness obtain crude product 1;
(7) crude product 1 is used dissolve with methanol, extremely use the good C-18 solid-phase extraction column of acetonitrile solution balance of volume ratio 25% with loading behind the membrane filtration of 0.45 μ m, be 5 bed volumes of 25% acetonitrile solution flushing with volume ratio again after sample adds, then use the acetonitrile solution wash-out of volume ratio 65%, collect elution peak, concentrated evaporate to dryness obtains crude product 2;
(8) with crude product 2 usefulness dissolve with methanol, the centrifugal 10min of 12000rpm, and after crossing 0.45 μ M filter membrane, separate with preparation HPLC; A liquid is the aqueous solution that contains volume ratio 0.1% trifluoroacetic acid after separating, and B liquid is trifluoroacetic acid aqueous solution; Gradient is: 40min in the time B liquid bring up to 60% from volume ratio 15%; Collect active part, 55～65 ℃ of concentrated ,-50 ℃～-60 ℃ vacuum lyophilizations namely get product cyclic lipopeptide microbiotic.
Described fermention medium is: glucose, 3.5g/L; CaCl
2, 0.05g/L; MgSO
47H
2O, 0.1g/L; ZnSO
4, 0.01g/L; NaCl, 0.3g/L; FeSO
4, 0.01g/L; (NH
4)
2SO
4, 2.5g/L; MnSO
4H
2O, 0.004g/L; KH
2PO
4, 2.5g/L; Solvent is water, and pH 7.0～7.2.
6, cyclic lipopeptide Antibiotic use is characterized in that: the cyclic lipopeptide microbiotic can be used in the preparation bacterial-infection resisting medicine.
The cyclic lipopeptide microbiotic is for the preparation of the medicine that infects due to the anti-multidrug resistant bacterium.The cyclic lipopeptide microbiotic is for the preparation of the medicine that infects due to the anti-multidrug resistant Gram-negative bacteria.
Preferred described macroporous adsorbent resin is Amberlite XAD-16, and solid-phase extraction column is C-18.Macroporous adsorbent resin has very strong specific adsorption performance, is widely used in the extraction of the materials such as microbiotic, VITAMIN and Effective Component of Chinese Medicine, concentrated and purifying.Macroporous adsorbent resin is generally the White-opalescent spheroid, has very strong absorption for microbiotic and small-molecule peptide, can be efficiently the specific target compound that is purified into from bulk fermentation liquid/extracting solution fast.In addition, macroporous adsorbent resin also can be used for removing non-polar compound from polar solvent.In a word, the absorption property of macroporous adsorbent resin is good, is a kind of desirable Solid-Phase Extraction material.
The present invention has following advantage: the invention provides a kind of toxicity and be better than PXB, have ring nonapeptide rhzomorph of higher medical use value and its preparation method and application, this compound is to all Gram-positives of testing and negative pathogenic bacterium and fungi, comprise multidrug resistant and super resistant organism, all have significant restraining effect; Experiment shows that this compound has obvious prevention effect to the mouse septicemia that the false unit cell of super wide spectrum resistance verdigris causes in the body.
Description of drawings
The ESI-MS collection of illustrative plates of the cyclic lipopeptide antibiosis prime ring nonapeptide rhzomorph of Fig. 1 embodiment 1 preparation.
The ESI-MS-MS collection of illustrative plates of the cyclic lipopeptide antibiosis prime ring nonapeptide rhzomorph of Fig. 2 embodiment 1 preparation.
Embodiment
Embodiment 1: the preparation of cyclic lipopeptide antibiosis prime ring nonapeptide rhzomorph
(1) bacterial strain B7 is transferred on the nutrient agar from the glycerine pipe that is stored in-80 ℃ of refrigerators, cultivates 24～72h for 32 ℃;
(2) will activate the bacterial strain access and be contained in the 50mL seed culture medium of 250mL triangular flask, in 32 ℃, 220rpm shaking culture 24h is primary seed solution;
(3) the primary seed solution access is contained in the 200mL seed culture medium of 500mL triangular flask, in 32 ℃, 220rpm shaking culture 24h is secondary seed solution;
(4) secondary seed solution is contained in the 2L triangular flask of 500mL fermention medium by the access of 5%～10%(v/v) inoculum size, in 28 ℃, 200rpm shaking culture 96h obtains fermented liquid.
Nutrient agar is prepared by following composition: extractum carnis 3g, peptone 10g, sodium-chlor 5g, agar 17g, distilled water 1000mL, pH7.2;
Seed culture medium is nutrient broth medium, prepares by following composition: extractum carnis 3g, peptone 10g, sodium-chlor 5g, tap water 1000mL, pH7.2;
Fermention medium is prepared by following composition: glucose, glucose, 3.5g/L; CaCl
2, 0.05g/L; MgSO
47H
2O, 0.1g/L; ZnSO
4, 0.01g/L; NaCl, 0.3g/L; FeSO
4, 0.01g/L; (NH
4)
2SO
4, 2.5g/L; MnSO
4H
2O, 0.004g/L; KH
2PO
4, 2.5g/L; Tap water complements to 1L, and pH 7.0.
(5) fermented liquid forwards in the centrifugal bottle, in the centrifugal 30min of 6500 * g, removes precipitation after the balance, gets the clarification fermented supernatant fluid;
(6) supernatant liquor is worn the good Amberlite XAD-16 chromatography column of distilled water balance with constant flow velocity stream, on sample complete rear respectively with distilled water and contain 45%(v/v) aqueous solution of methyl alcohol respectively washes 5 bed volumes, then with containing 90%(v/v) aqueous solution wash-out of methyl alcohol, collect the elution peak of 90% methanol aqueous solution wash-out, concentrated evaporate to dryness is designated as crude product 1.
(7) crude product 1 is used dissolve with methanol, with loading behind the membrane filtration of 0.45 μ m to using 25%(v/v) C-18 solid-phase extraction column that the acetonitrile solution balance is good, use again 25%(v/v after sample adds) 5 bed volumes of acetonitrile solution flushing, then use 65%(v/v) the acetonitrile solution wash-out, collect elution peak, concentrated evaporate to dryness is designated as crude product 2.
(8) with crude product 2 usefulness dissolve with methanol, centrifugal (12000rpm, 10min), and after crossing 0.45 μ M filter membrane, separate with preparation HPLC.A liquid is for containing 0.1%(v/v) aqueous solution of trifluoroacetic acid, B liquid is trifluoroacetic acid aqueous solution, gradient is: 40min in the time B liquid from 15%(v/v) bring up to 60%.Collect active part, concentrated, vacuum lyophilization namely get the product sterling.
The mensuration of embodiment 2 cyclic lipopeptide antibiosis prime ring nonapeptide rhzomorphs
This compound cyclic lipopeptide antibiosis prime ring nonapeptide rhzomorph correlation parameter of product of embodiment 1 preparation is as follows:
Outward appearance: be white powder after the lyophilize;
Molecular weight: 1100;
Molecular formula: C
54H
92N
12O
12
ESI-MS collection of illustrative plates: see Fig. 1;
ESI-MS-MS collection of illustrative plates: see Fig. 2.
According to above-mentioned characteristic, this compound of deducibility is a kind of nine amino acid whose new cyclic lipopeptide microbiotic that contain, with its called after ring nonapeptide rhzomorph.This microbiotic is the another new cyclic lipopeptide microbiotic that is separated to from series bacillus first both at home and abroad.This compound all has significant restraining effect to all Gram-positives of testing (staphylococcus) and feminine gender (pseudomonas and colon bacillus) pathogenic bacterium and fungi (Candida albicans).Experiment shows that this compound has good provide protection to the mouse infection due to the multi-resistant Pseudomonas aeruginosa in the Mice Body.
Embodiment 3 cyclic lipopeptide antibiosis prime ring nonapeptide rhzomorph antibacterial activity in vitro are measured
Be the ring nonapeptide microbiotic of 256 μ g/mL with Mueller Hinton broth culture compound concentration, adopt doubling dilution will encircle nonapeptide concentration and be transferred to respectively 256,128,64,32,16,8,4,2,1,0.5 μ g/mL.
In the 1st to the 10th hole of 96 hole polystyrene plates, add respectively the ring nonapeptide antibiotic solution behind the 50 μ L doubling dilutions, add 50 μ L Mueller Hinton broth culture (acid hydrolyzed casein 17.5g/L in the 11st hole, beef extract powder 2g/L, starch 1.5g/L) as growth control, adds 100 μ L Mueller Hinton broth cultures in the 12nd hole as negative control.The contrast microbiotic is PXB.
Indicator is diluted to 10 with Mueller Hinton broth culture after cultivating 24h
6Individual/mL, add 50 μ L dilution bacterium liquid in the to the 1st to the 11st hole, seal 37 ℃ of cultivation 20h in the rearmounted incubator, judged result.At this moment, the concentration of the 1st to the 10th hole Chinese style (I) new compound is respectively 128,64,32,16,8,4,2,1,0.5,0.25 μ g/mL.
Measure the OD value with microplate reader, the detection wavelength is 600nm.The minimum concentration that suppresses the indicator growth fully is defined as minimum inhibitory concentration (MIC).
The results are shown in Table 1.
Escherichia coli 5539 is the clinical multiple antibiotic resistant strain that separates with Pseudomonas aeruginosa 5215, wherein P.aeruginosa 5215 is general resistant organism bacterial strain, and is namely all insensitive to all common antibiotics of testing (Amikacin Sulphate, cefepime, imipenum, Cefuroxime sodium, cefotaxime, Ampicillin Trihydrate, Kefzol, cefoxitin, Levofloxacin, cefuroxime axetil, ceftazime, gentamicin, Meropenem, Sulbactam/Cefoperazone and piperacillin/his azoles clings to).The lipopeptide antibiotics PXB similar with ring nonapeptide rhzomorph has anti-microbial effect to gram-negative bacterias such as Pseudomonas aeruginosa, intestinal bacteria, bacillus canalis capsulatuses.Be mainly used in clinically the severe infections due to aminoglycoside-resistant, anti-third generation cephalosporin bacterium and Pseudomonas aeruginosa or other sensitive organism, such as microbemia, endocarditis, pneumonia, burn postoperative infection etc.As shown in Table 1, the new lipopeptide antibiotic of the present invention all shows stronger bacteriostatic activity to all Gram-positives of testing and negative pathogenic bacterium, wherein fungistatic effect and the PXB to Pseudomonas aeruginosa is suitable, to the fungistatic effect of escherichia coli a little less than PXB, but 3 strain staphylococcuses and Candida albicans to test have all shown more significant antibacterial effect, and prompting ring nonapeptide rhzomorph has wider antimicrobial spectrum.
Table 1: ring nonapeptide rhzomorph and PXB antibacterial activity in vitro
PXB toxicity is larger, has limited the effect of its clinical application.Ring nonapeptide rhzomorph is to (iv) LD of the intravenous injection of Kunming mouse
50Be 23.36mg/kg, apparently higher than PXB (7.35mg/kg), show that the toxicity of ring nonapeptide rhzomorph provided by the invention significantly is lower than the PXB of clinically widespread use, security is better.
In vivo in the Protection; the ring nonapeptide microbiotic of intravenously administrable 4mg/kg has 90% protection effect to the fatal infection of the false unit cell 5215 of super wide spectrum resistant organism verdigris; this shows that ring nonapeptide rhzomorph also has obvious antibacterial effect in animal body, can be applicable to prepare bacterial-infection resisting medicine.
The anxious poison experiment of embodiment 4 mouse
1, mouse
Mouse is the Kunming kind, in 6～8 weeks, weighs 18～22g.Processing to mouse is all carried out with reference to laboratory animal nursing and instruction manual (National Academy Press 1996).When finding that mouse is in dying state, adopt cervical vertebra dislocation execution method to alleviate the misery of mouse.Mouse is provided by Shanghai Si Laike laboratory animal responsibility company limited.
2, experimental technique
2.1 dosage setting: 40mg/kg, 32mg/kg, 25.6mg/kg, 20.5mg/kg, five groups of 16.38mg/kg.
2.2. test key step: adopt the test of Bliss method, get the kunming mice random packet, every group of 20 mouse, male and female half and half.Adopt the method for vein single-dose to mouse tail vein injection, volume injected is 0.5mL/20 gram mouse.Injection speed is strict controlled in 1mL/1 minute.Observe immediate reaction, statistics animal dead number.
3, experimental result
Ring nonapeptide rhzomorph vein single-dose is 23.36mg/kg to Kunming mouse acute toxicity LD50.Buck LD50 is 23.47mg/kg, and jenny LD50 is 23.24mg/kg, there was no significant difference between female tom.The LD50 of control drug PXB is 7.35mg/kg.
Embodiment 5: Mouse Protection Test
1, Pseudomonas aeruginosa 5215 best infection concentration trial tests
Get 25 of ICR mouse, male, body weight 18-22g is divided into 5 groups at random, and 5 every group, abdominal injection is pressed 5 kinds of concentration (2.5 * 10 of 1:10 proportional diluted with 1% sterilised yeast suspension respectively
7, 2.5 * 10
6, 2.5 * 10
5, 2.5 * 10
4, 2.5 * 10
3Individual/mL) Pseudomonas aeruginosa 5215 bacterium liquid 0.5mL/ only, the death condition of record in one week the results are shown in Table 2.Determine thus official test employing 2.5 * 10
6The Pseudomonas aeruginosa bacterium liquid of individual/mL infects.
The table 2 Pseudomonas aeruginosa 5215 best concentration trial test results that infect
2, ring nonapeptide rhzomorph is on the impact of abdominal cavity infection Pseudomonas aeruginosa 5215 mouse death rates
Get 50 of ICR mouse, body weight 18～22g, male, the Pseudomonas aeruginosa 5215 suspensions (concentration 2.5 * 10 that abdominal injection dilutes with 1% sterilised yeast suspension
6Individual/as ml), to be divided at random control group, PXB 2mg/kg positive controls, nonapeptide rhzomorph 1,2,4mg/kg Three doses group, 10 every group, intravenous injection gives relative medicine respectively.Administering mode is respectively every day 1 time, for three days on end.Each administration volume is the 0.2mL/10g body weight, and control group gives equal-volume physiological saline; The death condition of mouse in one week respectively organized in record, calculates mortality ratio, the results are shown in Table 3.
The result shows that ring nonapeptide rhzomorph all can make the mortality ratio of infecting mouse obviously reduce (compare with control group P＜0.01) with 2mg/kg and above dosed administration, shows that this dosage range has obvious vivo bacteria corrosion action to Pseudomonas aeruginosa.
Table 3: ring nonapeptide rhzomorph is on the impact of abdominal cavity infection Pseudomonas aeruginosa mouse death rate
Annotate: carry out X with the accurate stochastic method of Fisher
2Check,
*P＜0.01(and control group are relatively).
Claims (8)
1. cyclic lipopeptide microbiotic, it is characterized in that: skeleton symbol is as follows:
Wherein L-Dab is the L-type 2,4-diamino-butanoic, and L-Ile is the L-type Isoleucine, and D-Phe is D type phenylalanine, and L-Leu is the L-type leucine, and D-Val is D type α-amino-isovaleric acid, and L-Thr is the L-type Threonine.
2. a strain prepares the antibiotic bacterial strain of cyclic lipopeptide, it is characterized in that: described bacterial strain is series bacillus (Paenibacillus ehirnensis strain B7), be preserved in Chinese Typical Representative culture collection center, preservation date: on June 24th, 2012, the address: Wuhan, China Wuhan University, preserving number is CCTCC NO:M 2012244.
3. the antibiotic preparation method of cyclic lipopeptide is characterized in that step is:
(1) cultivation and fermentation: series bacillus (Paenibacillus ehirnensis strain B7) is inoculated into fermention medium, obtain activating bacterial strain, activation bacterial strain twice of shaking culture in seed culture medium, obtain secondary seed solution, shaking culture fermentation in the secondary seed solution access fermention medium, obtain fermented liquid, centrifugal, get fermented liquid supernatant, go precipitation, through absorption with macroporous adsorbent resin, the fermentation supernatant is flowed through with distilled water balance chromatography column good, that be filled with macroporous adsorbent resin with appropriate speed first;
(2) separation and purification: after complete on the sample, respectively with distilled water and methanol aqueous solution flushing, then use the methanol aqueous solution wash-out first; Collect each elution peak, survey and live, the concentrated evaporate to dryness of active ingredient is obtained crude product; With the crude product dissolve with methanol, centrifuging is further purified with solid-phase extraction column; Carry out consummately with preparative high performance liquid chromatography again, collect active ingredient, concentrated, freeze-drying can obtain described ring nonapeptide rhzomorph.
4. the antibiotic preparation method of cyclic lipopeptide as claimed in claim 3 is characterized in that concrete steps are:
The series bacillus (Paenibacillus ehirnensis strain B7) that (1) will be stored in the glycerine pipe of-80 ℃ of refrigerators is transferred on the nutrient agar, cultivates 24～96h, obtains activating bacterial strain for 28～35 ℃;
Nutrient agar is prepared by following composition: extractum carnis 3g, peptone 10g, sodium-chlor 5g, agar 17g, distilled water 1000mL, pH7.2;
(2) will activate the bacterial strain access and be contained in the 50mL seed culture medium of 250mL triangular flask, in 28～35 ℃, 220rpm shaking culture 24h is primary seed solution;
(3) the primary seed solution access with step (2) gained is contained in the 200mL seed culture medium of 500mL triangular flask, and in 28～35 ℃, 220rpm shaking culture 24h is secondary seed solution;
Seed culture medium is nutrient broth medium, prepares by following composition: extractum carnis 3g, peptone 10g, sodium-chlor 5g, tap water 1000mL, pH7.2;
(4) by volume 5%～10% the inoculum size access of secondary seed solution with step (3) gained contains in the 2L triangular flask of 500mL fermention medium, and in 28～35 ℃, 200rpm shaking culture 96h obtains fermented liquid;
Fermention medium is prepared by following composition: glucose, 0～10g/L; CaCl
2, 0.01～0.7g/L; MgSO
47H
2O, 0.05～1g/L; ZnSO
4, 0.005～0.1g/L; NaCl, 0.05～0.5g/L; FeSO
4, 0.005～0.05g/L; (NH
4)
2SO
4, 0.5～4.0g/L; MnSO
4H
2O, 0.001～0.01g/L; KH
2PO
4, 0.5～5g/L; Solvent is distilled water, and pH 7.0～7.5;
(5) fermented liquid obtained of step (4) forwards in the centrifugal bottle, in the centrifugal 30min of 6500 * g, removes precipitation after the balance, gets the clarification fermented supernatant fluid;
(6) supernatant liquor of step (5) gained is worn the good Amberlite XAD-16 chromatography column of distilled water balance with the flow velocity stream of 10～50mL/min, complete rear respectively with distilled water and to contain volume ratio be that the aqueous solution of 45% methyl alcohol respectively washes 5 bed volumes on sample, then with containing the aqueous solution wash-out that volume ratio is 90% methyl alcohol, collected volume is than the elution peak that is 90% methanol aqueous solution wash-out, 55～65 ℃ of concentrated evaporates to dryness obtain crude product 1;
(7) crude product 1 is used dissolve with methanol, extremely use the good C-18 solid-phase extraction column of acetonitrile solution balance of volume ratio 25% with loading behind the membrane filtration of 0.45 μ m, be 5 bed volumes of 25% acetonitrile solution flushing with volume ratio again after sample adds, then use the acetonitrile solution wash-out of volume ratio 65%, collect elution peak, concentrated evaporate to dryness obtains crude product 2;
(8) with crude product 2 usefulness dissolve with methanol, the centrifugal 10min of 12000rpm, and after crossing 0.45 μ M filter membrane, separate with preparation HPLC; A liquid is the aqueous solution that contains volume ratio 0.1% trifluoroacetic acid after separating, and B liquid is trifluoroacetic acid aqueous solution; Gradient is: 40min in the time B liquid bring up to 60% from volume ratio 15%; Collect active part, 55～65 ℃ of concentrated ,-50 ℃～-60 ℃ vacuum lyophilizations namely get product cyclic lipopeptide microbiotic.
5. the antibiotic preparation method of cyclic lipopeptide as claimed in claim 4, it is characterized in that: described fermention medium is: glucose, 3.5g/L; CaCl
2, 0.05g/L; MgSO
47H
2O, 0.1g/L; ZnSO
4, 0.01g/L; NaCl, 0.3g/L; FeSO
4, 0.01g/L; (NH
4)
2SO
4, 2.5g/L; MnSO
4H
2O, 0.004g/L; KH
2PO
4, 2.5g/L; Solvent is water, and pH 7.0～7.2.
6. cyclic lipopeptide Antibiotic use, it is characterized in that: the cyclic lipopeptide microbiotic can be used in the preparation bacterial-infection resisting medicine.
7. cyclic lipopeptide Antibiotic use as claimed in claim 6 is characterized in that: the cyclic lipopeptide microbiotic is for the preparation of the medicine that infects due to the anti-multidrug resistant bacterium.
8. cyclic lipopeptide Antibiotic use as claimed in claim 7 is characterized in that: the cyclic lipopeptide microbiotic is for the preparation of the medicine that infects due to the anti-multidrug resistant Gram-negative bacteria.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210356785.7A CN102911257B (en) | 2012-09-21 | 2012-09-21 | Cyclic lipopeptide antibiotic and preparation and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210356785.7A CN102911257B (en) | 2012-09-21 | 2012-09-21 | Cyclic lipopeptide antibiotic and preparation and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102911257A true CN102911257A (en) | 2013-02-06 |
| CN102911257B CN102911257B (en) | 2014-02-12 |
Family
ID=47609857
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210356785.7A Expired - Fee Related CN102911257B (en) | 2012-09-21 | 2012-09-21 | Cyclic lipopeptide antibiotic and preparation and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102911257B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104561235A (en) * | 2013-10-22 | 2015-04-29 | 上海天伟生物制药有限公司 | Sterile detection method |
| CN106467922A (en) * | 2016-08-26 | 2017-03-01 | 上海交通大学 | A kind of synthetic method of the lipopeptid based on Pseudomonas chlororaphis |
| CN109666604A (en) * | 2018-12-24 | 2019-04-23 | 光明乳业股份有限公司 | A kind of highly concentrated lactobacillus plantarum multiplication agent and preparation method thereof and application method |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1616484A (en) * | 2004-09-27 | 2005-05-18 | 沈阳药科大学 | Novel cyclopeptide anti-tumor, anti-virus antibiotic active compound |
| CN101519435A (en) * | 2009-04-09 | 2009-09-02 | 中国人民解放军第二军医大学 | Cyclic nonapeptide compound in Hsisha sponge and application thereof |
| CN101962401A (en) * | 2010-09-01 | 2011-02-02 | 浙江大学 | Polypeptin and preparation and application thereof |
| CN102030819A (en) * | 2010-11-22 | 2011-04-27 | 浙江大学 | Broad spectrum polypeptin and application thereof |
| CN102432674A (en) * | 2010-09-29 | 2012-05-02 | 上海天伟生物制药有限公司 | Method for purifying cyclic lipopeptide compound or salt thereof |
-
2012
- 2012-09-21 CN CN201210356785.7A patent/CN102911257B/en not_active Expired - Fee Related
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1616484A (en) * | 2004-09-27 | 2005-05-18 | 沈阳药科大学 | Novel cyclopeptide anti-tumor, anti-virus antibiotic active compound |
| CN101519435A (en) * | 2009-04-09 | 2009-09-02 | 中国人民解放军第二军医大学 | Cyclic nonapeptide compound in Hsisha sponge and application thereof |
| CN101962401A (en) * | 2010-09-01 | 2011-02-02 | 浙江大学 | Polypeptin and preparation and application thereof |
| CN102432674A (en) * | 2010-09-29 | 2012-05-02 | 上海天伟生物制药有限公司 | Method for purifying cyclic lipopeptide compound or salt thereof |
| CN102030819A (en) * | 2010-11-22 | 2011-04-27 | 浙江大学 | Broad spectrum polypeptin and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| SOGN J.: "Structure of the Peptide Antibiotic Polypeptin", 《JOURNAL OF MEDICINAL CHEMISTRY》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104561235A (en) * | 2013-10-22 | 2015-04-29 | 上海天伟生物制药有限公司 | Sterile detection method |
| CN106467922A (en) * | 2016-08-26 | 2017-03-01 | 上海交通大学 | A kind of synthetic method of the lipopeptid based on Pseudomonas chlororaphis |
| CN106467922B (en) * | 2016-08-26 | 2021-04-02 | 上海交通大学 | Synthesis method of lipopeptide based on pseudomonas chlororaphis |
| CN109666604A (en) * | 2018-12-24 | 2019-04-23 | 光明乳业股份有限公司 | A kind of highly concentrated lactobacillus plantarum multiplication agent and preparation method thereof and application method |
| CN109666604B (en) * | 2018-12-24 | 2022-05-06 | 光明乳业股份有限公司 | Highly concentrated lactobacillus plantarum proliferation agent and preparation method and use method thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102911257B (en) | 2014-02-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102871996A (en) | Antibiotic composition and application thereof | |
| CN101240013B (en) | High purity lipopeptides, lipopeptide micelles, processes for preparing same | |
| Nagoba et al. | Medical applications of siderophores–a review | |
| Chavan et al. | Antibacterial and antifungal drugs from natural source: A review of clinical development | |
| US9849145B2 (en) | Pharmaceutical composition containing honeysuckle extract and antibiotics, pharmaceutical kit, and use of honeysuckle extract for preparation of drug | |
| JP2014518878A (en) | Synergistic combinations of polyene fungicides and non-ribosomal peptides and related methods of use | |
| CA3053850A1 (en) | Antifungal agents and uses thereof | |
| CN102911257B (en) | Cyclic lipopeptide antibiotic and preparation and application thereof | |
| CN111518163B (en) | Application of lipopeptide compounds in resisting novel coronavirus | |
| CN114209808B (en) | Application of polypeptide RK12 in preparation of medicines for treating acne | |
| CN103920137B (en) | A kind of pharmaceutical composition with the effect of anti-drug resistance gram-positive bacteria | |
| EP0563844B1 (en) | Antimicrobial compositions and pharmaceutical preparations thereof | |
| CN102558307B (en) | Octapeptin as well as preparation and application thereof | |
| CN101019875A (en) | Application of sophorolipid in preparing antibiotic medicine | |
| CN103142993A (en) | Application of Anoplin in treatment of liver cancer | |
| US11718585B2 (en) | Bis-cyclic guanidines as antibacterial agents | |
| Schwan | Mushrooms: an untapped reservoir for nutraceutical antibacterial applications and antibacterial compounds | |
| CN111494364B (en) | Application of isopentenyl substituted phenol compound in resisting staphylococcus aureus and methicillin-resistant staphylococcus aureus | |
| CN109678944A (en) | A kind of antibacterial polypeptide HF-18 and its preparation method and application | |
| CN110283237A (en) | There is antimicrobial function peptidomimetic and preparation method thereof, composition and application from disease fungus | |
| Thornton et al. | Laboratory and clinical studies of a new antibiotic, cephaloridine, in the treatment of gram-positive infections. | |
| JP2005524622A (en) | Dab9 derivatives of lipopeptide antibiotics and methods of making and using the same | |
| RU2661613C1 (en) | Method for treatment of human diseases caused by gram-positive bacteria | |
| CN103396471A (en) | Metal beta-lactamase inhibitory peptide and application thereof | |
| CN111909243B (en) | Antibacterial peptide, antibacterial and itching-relieving pharmaceutical composition and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20140212 Termination date: 20150921 |
|
| EXPY | Termination of patent right or utility model |