CN109453185A - New application of the aurantiamarin as metallo-β-lactamase inhibitor - Google Patents
New application of the aurantiamarin as metallo-β-lactamase inhibitor Download PDFInfo
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- CN109453185A CN109453185A CN201811606283.9A CN201811606283A CN109453185A CN 109453185 A CN109453185 A CN 109453185A CN 201811606283 A CN201811606283 A CN 201811606283A CN 109453185 A CN109453185 A CN 109453185A
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- aurantiamarin
- metallo
- lactamase
- antibiotic
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
- A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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Abstract
The invention discloses a kind of new applications of aurantiamarin; it can be used as metallo-β-lactamase inhibitor; for preventing the compound sensitive to metallo-β-lactamase from being digested, hydrolysis of the metallo-β-lactamase to beta-lactam antibiotic is especially prevented, is played a protective role to such antibiotic.
Description
Technical field
The present invention relates to a kind of new applications of aurantiamarin, that is, metallo-β-lactamase inhibitor are used as, for preventing metal
Hydrolysis of the beta-lactamase to beta-lactam antibiotic, plays a protective role to such antibiotic.Its application is included in mould
Aurantiamarin is added in all kinds of preparations of the beta-lactam antibiotics such as element, to inhibit the microorganism for producing metallo-β-lactamase to β-
The hydrolysis of lactam antibiotics maintains the curative effect of antibiotic to improve the stability of beta-lactam antibiotic.
Background technique
Beta-lactam antibiotic good effect and small toxicity are the important drugs of current treatment infectious diseases.Such medicine
Object includes penicillins, cephalosporins, penems, monobactam class etc., and all there is one to play in their structures and resist
The indispensable beta-lactam nucleus of bacterium activity.
Beta-lactamase is the bacteriogenic resistance to drug metabolizing enzyme of one kind, it can be catalyzed the lactam nucleus in beta-lactam antibiotic
Hydrolysis and destroy, thus make bacterium to beta-lactam antibiotic generate drug resistance.Based on DNA sequence dna similitude, β-is interior
Amidase is divided into 4 classes: A-D.Wherein, tri- fermentoid of A, C, D plays catalytic action dependent on the Ser in activated centre, can be by carat
The antibiotic that dimension acid, Tazobactam Sodium and Sulbactam etc. clinically use is inhibited；And B fermentoid needs to rely on the Zn hair in activated centre
Catalytic action enzyme is waved, referred to as metallo-β-lactamase (MBL), fermentoid clinically there is no effective inhibitor.
On August 11st, 2010 Britain " lancet-infectious disease " periodicals published report a kind of new superbacteria, the bacterium
NDM-1 type metallo-β-lactamase (New Delhi metallo-β-lactamase 1) can be generated, the novel bacterial of this drug-resistance factor is carried
All there is drug resistance to nearly all antibiotic almost including carbapenem, mortality is very high.Since this is resistance to
The gene of the medicine factor can independently replicate, and can be transferred to different genera there are on plasmid independently of except chromosome
Bacterium in, germ possesses the surprising potential of propagation and variation.The appearance of NDM-1 superbacteria causes the concern in the whole world and tight
, the World Health Organization calls upon States to reinforce monitoring, and various countries is urged to take measures to resist drug tolerant bacteria, actively reduces bacterium pair
Antibiotic generates the chance of anti-medicine.The appearance of NDM-1 is but also the research of metallo-β-lactamase and its inhibitor becomes the whole world
The hot spot of concern.
Aurantiamarin is also known as hesperidin, hesperidin etc., is contained in the pericarps such as lemon, citrus, bitter orange flower, is a kind of flavonoid glycosides
Compound.It has citrin sample efficiency, with entering in skin-care products, plays the role of preventing frostbite.It can be anti-inflammatory and disease-resistant
Poison has certain antibacterial action, and use can inhibit the generation of dental plaque and reduce halitosis simultaneously in oral hygiene product.It can prevent
Action of UV radiation can be used for sunscreen products.But aurantiamarin is had not yet to see as MBL inhibitor, especially to super thin
Report in terms of the inhibitory activity for the NDM-1 type metallo-β-lactamase that bacterium generates, this patent has found aurantiamarin, and this is new
Activity can be used for inhibiting to produce hydrolysis of the MBL microorganism to beta-lactam antibiotic, the stability of antibiotic be improved, as β-
The potential drug of the protective agent of lactam antibiotics and anti-superbacteria, and with good development and application prospects.
Summary of the invention
The present invention relates to the new function of aurantiamarin and its applications, it can inhibits metallo-β-lactamase, for improving β-
The stability of lactam antibiotics extends action time, improves the activity of antibiotic.
According to an aspect of the invention, there is provided metallo-β-lactamase inhibitor comprising by following structural formula table
The compound shown or its solvate:
The compound of the present invention source is simple, and raw material can be extracted from plants, and can also purchase from raw material commercial source
It can buy.
The invention discloses aurantiamarins as the application in terms of metallo-β-lactamase inhibitor, and aurantiamarin can be by solid
The forms such as body powder, particle and solution are mixed with beta-lactam antibiotic, to protect antibiotic not by Metallo-β-lactamases
Enzyme hydrolysis improves the stability of beta-lactam antibiotic.
Here is detection of the aurantiamarin to NDM-1 type metallo-β-lactamase inhibitory activity, uses 60 μM in reaction process
Cefuroxime Sodium is substrate, and buffer used is 50mM HEPES (4- hydroxyethyl piperazineethanesulfonic acid), 200mM NaCl, 100 μM
ZnCl2, pH=7.2, reaction temperature is 30 DEG C, the final concentration of 10nM of the metallo-β-lactamase used, and Detection wavelength is
260nm, detailed process is as follows:
1. metallo-β-lactamase to be configured to the solution of concentration 10nM with buffer first, 30 DEG C of incubation 10min are placed in, so that
Zn2+Sufficiently occupy activated centre；
2. aurantiamarin is dissolved in buffer, it is configured to different concentration, is added in enzyme solution, 30 DEG C of incubation 6min make to inhibit
Agent is sufficiently combined with enzyme；
3. the above-mentioned solution of 1ml is taken to be added in quartz colorimetric utensil, 3 μ L Cefuroxime Sodiums (60 μM of final concentration) is added and shakes up, surveys immediately
Determine the variation of system absorbance, records data.
Since substrate Cefuroxime Sodium is declined by its absorbance after enzyme hydrolysis, thus can be with by the variation of measurement absorbance
Characterize the hydrolysis degree of substrate.Inhibiting rate of the aurantiamarin to β-lactamase hydrolysis substrate of various concentration is calculated, with compound
Concentration to inhibiting rate matched curve, calculate IC50Value, the results showed that aurantiamarin inhibits NDM-1 type metallo-β-lactamase
IC50It is 3.348 ± 1.35 μM.
Embodiment
The beneficial effect that come that the present invention is further explained by the following examples and to the test of the Vaccine effectiveness of antibiotic described
Fruit
Test production MBLs bacterial strain used is genetically engineered E.coli BL21 (DE3)/pET28a-NDM-1 bacterium, the bacterium
Strain can be hydrolyzed beta-lactam antibiotic, inactivate antibiotic with solubility expression NDM-1 type beta-lactamase intracellular.With
E.coli BL21 (DE3)/pET28a bacterial strain is as negative control.It is measured using doubling dilution, orange is reflected by protective index
Protective effect of the skin glycosides to antibiotic.
Protective index is defined as, and when reaching identical active effect (such as antibacterial), antibiotic is used when being not added with protective agent
Concentration, with addition protective agent when antibiotic used in concentration ratio, such as following formula: the value be greater than 1 when, show be added protective agent
Afterwards, antibiotic needs amount to be used that can reduce, and the value is bigger, shows that protectant effect is stronger.
The specific method is as follows:
By the engineering bacterial strain E.coli BL21 (DE3) saved in ultra low temperature freezer /pET28a-NDM-1 (zymogenic bacteria) with
And control strain E.coli BL21 (DE3)/pET28a (control bacterium) is inoculated in the Luria-Bertani (LB) of sterilizing admittedly respectively
In body culture medium, culture 12 hours is inverted in 37 DEG C of constant incubators.It is transferred to sterilizing respectively with oese picking single colonie
LB liquid medium in, in 37 DEG C, shake culture is to logarithmic growth phase under the conditions of 200rpm.It is measured with ultraviolet specrophotometer
Absorbance value (the OD of bacterium solution at 600nm wavelength600), according to 1OD=1 × 109The conversion relation of CFU/mL, respectively will expression gold
The engineering bacterial strain and control strain of category beta-lactamase are diluted to 1 × 10 with sterile LB medium6CFU/mL is added to nothing
In 96 orifice plate of bacterium, antibiotic, antibiotic+aurantiamarin is used alone to the rejection ability of bacterium in measurement respectively, measures aurantiamarin pair
The protective index of antibiotic.
Embodiment one:
Precision weighs aurantiamarin 32mg, and the distilled water 10mL of sterilizing is added, and dissolves by heating and is used as stock solution.By antibiotic head
Spore cefuroxime sodium sterile saline (0.9%NaCl) dissolution is configured to 512 μ g/mL solution, and 100 μ l is taken to be added to 96 orifice plates
In 1st column hole, then from taking 50 μ l to be transferred in the 2nd column hole in the hole, 50 μ l of sterile saline is added, is carefully blown with liquid-transfering gun
Suction mixes several times, then 50 μ l is taken to be transferred in the 3rd column hole, and sterile saline is added with method and carries out two doubling dilutions.Then each
It is separately added into 50 μ l bacterium solutions in hole, makes final concentration of 256~1 μ g/mL of antibiotic.Test group is separately added into 1 μ l aurantiamarin deposit
Liquid, the corresponding final concentration of 32 μ g/mL of aurantiamarin；1 μ l sterile distilled water is added in control group.The above each group is in 37 DEG C of constant incubators
Culture 12-16 hours, observes the growing state of each hole bacterium, and the results are shown in Table 1, and wherein "+" represents bacterium growth, and "-" represents
Bacterium does not grow:
Protective index measurement result of 1. aurantiamarin of table to Cefuroxime Sodium
It can be obtained from 1 result of table, Cefuroxime Sodium is used alone to the E.coli bacterium for producing NDM-1 type metallo-β-lactamase
MIC is 128 μ g/mL, and after aurantiamarin is added, Cefuroxime Sodium is 64 μ g/mL to the MIC of the zymogenic bacteria；It is corresponding to be free of cephalo
Cefuroxime sodium is only added in the control group of 32 μ g/mL aurantiamarins, and drug-fast bacteria normal growth shows under the concentration, and orange is used alone
Skin glycosides is to the no inhibiting effect of the growth of zymogenic bacteria, and aurantiamarin is the hydrolysis by inhibiting NDM-1 to Cefuroxime Sodium, to increase
The strong anti-zymogenic bacteria activity of Cefuroxime Sodium.It is 2 to the protective index of Cefuroxime Sodium that aurantiamarin, which is calculated,.
Embodiment two:
Precision weighs aurantiamarin 24mg, and the distilled water 10mL of sterilizing is added, and dissolves by heating and is used as stock solution.By antibiotic beauty
Luo Peinan sterile saline (0.9%NaCl) dissolution is configured to 512 μ g/mL solution, and 100 μ l is taken to be added to the of 96 orifice plates
In 1 column hole, then from taking 50 μ l to be transferred in the 2nd column hole in the hole, 50 μ l of sterile saline is added, with the careful pressure-vaccum of liquid-transfering gun
It mixes several times, then 50 μ l is taken to be transferred in the 3rd column hole, sterile saline is added with method and carries out two doubling dilutions.Then in each hole
In be separately added into 50 μ l bacterium solutions, make final concentration of 256~1 μ g/mL of antibiotic.Test group is separately added into 1 μ l aurantiamarin stock solution,
The corresponding final concentration of 24 μ g/mL of aurantiamarin；1 μ l sterile distilled water is added in control group.The above each group is trained in 37 DEG C of constant incubators
It supports 12-16 hours, observes the growing state of each hole bacterium, the results are shown in Table 2, and wherein "+" represents bacterium growth, and "-" represents bacterium
It does not grow:
Protective index measurement result of 2. aurantiamarin of table to Meropenem
It can be obtained from 2 result of table, Meropenem is used alone to the E.coli bacterium MIC for producing NDM-1 type metallo-β-lactamase
For 64 μ g/mL, after aurantiamarin is added, Meropenem is 32 μ g/mL to the MIC of the zymogenic bacteria；It is corresponding to be free of Meropenem, only
It is added in the control group of 24 μ g/mL aurantiamarins, drug-fast bacteria normal growth shows under the concentration, and aurantiamarin is used alone to production
The no inhibiting effect of the growth of enzyme bacterium, aurantiamarin are the hydrolysis by inhibiting NDM-1 to Meropenem, to enhance Metro training
The anti-zymogenic bacteria activity in south.It is 2 to the protective index of Meropenem that aurantiamarin, which is calculated,.
Claims (5)
1. the purposes that aurantiamarin is used to prepare metallo-β-lactamase inhibitor.
2. the aurantiamarin in claim 1 includes its prototype, pharmaceutically acceptable salt and the preparation containing aurantiamarin.
3. the metallo-β-lactamase in claim 1 includes but is not limited to the common metals such as IMP-1, VIM-2 and NDM-1 β-
Lactamase.
4. the purposes in claim 1 is that it is anti-to be added to beta-lactam using aurantiamarin as metallo-β-lactamase inhibitor
In raw element, to inhibit the enzyme to the hydrolysis of antibiotic, play the role of protecting antibiotic.
5. the source of metallo-β-lactamase in claim 1, including extract from nature or prepared with genetic engineering bacterium.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811606283.9A CN109453185A (en) | 2018-12-24 | 2018-12-24 | New application of the aurantiamarin as metallo-β-lactamase inhibitor |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811606283.9A CN109453185A (en) | 2018-12-24 | 2018-12-24 | New application of the aurantiamarin as metallo-β-lactamase inhibitor |
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| Publication Number | Publication Date |
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| CN109453185A true CN109453185A (en) | 2019-03-12 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811606283.9A Pending CN109453185A (en) | 2018-12-24 | 2018-12-24 | New application of the aurantiamarin as metallo-β-lactamase inhibitor |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115869331A (en) * | 2023-01-04 | 2023-03-31 | 中国水产科学研究院黄海水产研究所 | Application of florfenicol and hesperidin composition in inhibition of vibrio parahaemolyticus |
-
2018
- 2018-12-24 CN CN201811606283.9A patent/CN109453185A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115869331A (en) * | 2023-01-04 | 2023-03-31 | 中国水产科学研究院黄海水产研究所 | Application of florfenicol and hesperidin composition in inhibition of vibrio parahaemolyticus |
| CN115869331B (en) * | 2023-01-04 | 2023-08-25 | 中国水产科学研究院黄海水产研究所 | Application of florfenicol and hesperidin composition in inhibiting Vibrio parahaemolyticus |
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| PB01 | Publication | ||
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Application publication date: 20190312 |