CN107519167B - Candida albicans resistant product combining harmine hydrochloride and fluconazole and application thereof - Google Patents
Candida albicans resistant product combining harmine hydrochloride and fluconazole and application thereof Download PDFInfo
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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
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
- A61K31/437—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a five-membered ring having nitrogen as a ring hetero atom, e.g. indolizine, beta-carboline
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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
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
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Abstract
The invention discloses an antifungal product combining harmine hydrochloride with fluconazole and application thereof, belongs to the technical field of medicines, and researches the combined antifungal effect of the combination of fluconazole and harmine hydrochloride on drug-resistant candida albicans by using various methods. The effective concentration ratio of the harmine hydrochloride and the fluconazole in the combined application is as follows: and (3) fluconazole: harmine hydrochloride =1:32 (μ g/mL for CA 3) or 1:16 (μ g/mL for CA 4), the minimum inhibitory concentrations being: 8 μ g/mL versus 0.25 μ g/mL (for CA 3) or 0.5 μ g/mL (for CA 4). When the harmine hydrochloride is combined with the fluconazole, the antibacterial activity of the fluconazole on the drug-resistant candida albicans can be enhanced, a synergistic antifungal effect can be generated, the drug resistance of the drug-resistant candida albicans on the fluconazole can be reversed, and a research direction is provided for the development of new drugs and the new application of old drugs.
Description
Technical field
The present invention relates to pharmaceutical technology fields, and in particular to the antifungal products of Harmine HCl joint Fluconazole
And its
Using.
Background technique
Candida albicans (Candida albicans, CA) is common opportunistic fungus, belongs to Saccharomycetaceae beads
Pseudomonas.Seldom there is systemic fungal infection in the normal situation of immune function of human body, but in Past 30 Years, due to immune
The appearance of a variety of predisposing factors such as inhibition, organ transplant, Immune Deficiency Syndrome, aggressive monilial infection
(invasive Candidia infection, ICI) rate is in continuous raised situation, seriously threatens the life security of patient.To the greatest extent
Pipe ICI as caused by various non-white candida albicans (non-Candida albicans) is risen, but current white candida albicans
It is still to lead to the most important pathogen of ICI.By taking the domestic a 2013 multicenter investigation results of study carried out as an example, by white
The 41.8% of the total patient's ICI number of ICI Zhan caused by color candida albicans, case fatality rate is still up to 36.6% after treatment patients.It is another
Part is also shown by the data that Chinese Hospitals invasive fungi detection net (CHIFNET) is newly issued, and Candida albicans, which accounts for, causes ICI sick
The 50.1% of opportunistic pathogen sum.
Drug combination is that antimycotic drug resistance drug combination research includes two kinds of antifungal joints and non-antifungal and resists
Fungi medicine joint, since current useful clinically antifungal drug kind is limited, and mostly it is expensive, toxic side effect is obvious,
So that the use in conjunction research of non-antifungal drug and antifungal drug is concerned.Fluconazole (fluconazole, FLC) is
Third generation triazole antifungal agent is widely used from after listing by good clinical efficacy, but long-term, high-dose use is led
Cause nosocomial infection drug-fast bacteria separation rate higher and higher, in the world the antimycotic monitoring network ARTEMIS project of maximum-norm
The data of (ARTEMIS program) publication in 2010 are shown, are clinically separated from all over the world from 2001 to 2007
Be more than out in 120,000 plants of Candida albicans discovery fluconazole resistant rate be 1.4%.However, in the resistance to of China's Candida albicans
Medicine rate, although lacking the investigation of national large area, has data to show compared with world average level height, at present China's Partial Hospitals
Candida albicans has been up to 10.4% to the resistant rate of Fluconazole, it is seen that Candida albicans drug-fast bacteria infection problem in nosocomial infection
Increasingly serious, this just needs the research for carrying out and reinforcing overriding resistance Candida albicans drug.Drug combination is that antimycotic resistance to have
Effect approach, the research of medicine drug combination mainly include that two kinds of antifungal joints and non-antifungal are combined with antifungal, due to
Current useful clinically antifungal drug kind is limited, and mostly it is expensive, toxic side effect is obvious, and find the increasing of Fluconazole
Quick dose has become research hotspot both domestic and external to overriding resistance Candida albicans.
Harmel (Peganum harmala L.) is perennial herb zygophyllaceae Peganum Three Plants, be Uygurs, China,
Mongols's medicinal herbs most in use are put into the Sanitation Ministry medicine standard Uygur nationality fascicle.It is distributed mainly on northwest China area, in
East, the Central Asia, Mediterranean, South America are also distributed.Harmel is mild-natured, bitter, pungent, toxic, has firm tendon and vessel, supporing yang warm
The functions such as yin, dissipation cold-dampness gas cure mainly tendon and vessel weakness, osteoarthrosis pain, coughing with a lot of sputum, hemiplegia amnesia, coma headache, irregular menstruation
Equal diseases.Seed and herb all have medical value, and one of main active is yageine (7- methoxyl group -1- first
Base -9H- pyrido [3,4-b] indoles, harmine, HAR).Yageine is also known as meat Ye Yunxiang alkali, faint yellow needle-shaped crystalline substance
Body, molecular weight 212.25, molecular formula Cl3H12N20, it is a kind of tricyclic beta-carboline alkaloid.Yageine is in nature
It is widely distributed in boundary, including various plants, marine organisms, insect etc., most earlier than 1874 by people from the seed of harmel
Extraction obtains.Its content in seed of peganum harmala is higher, stable structure.It is current studies have shown that Harmine HCl
With a series of extensive pharmacological actions such as antibacterium, antiplasmodial, antimycotic, anti-oxidant, antitumor.In Traditional Folk medication
On the experiential basis of accumulation, the research for effective monomer compound in medicinal plant is in widespread attention.Xu Yan et al. is used
Test tube method detects Harmine HCl to staphylococcus aureus, beta hemolytic streptococcus, Pseudomonas aeruginosa, large intestine bar
Bacterium, Salmonella typhi, the minimum bactericidal concentration (MBC) of Shigella flexneri and Candida albicans and to staphylococcus aureus, green
Purulence bacillus, Candida albicans time-killing curve.Test result: Harmine HCl is to the MBC of 6 kinds of bacteriums
125~250 μ g/mL, the MBC to Candida albicans are 800 μ g/mL.But Harmine HCl itself have it is certain
Toxicity, usage amount is excessively high to be caused maincenter illusion, trembles, decreased heart rate and arrhythmia, can also inhibit to breathe, it is serious even
Lead to death by suffocation, these are all an important factor for limiting its use.Therefore, the usage amount for reducing Harmine HCl exists
Have great importance in medical research.Whether there is the anti-white of collaboration for the combination of Harmine HCl and Fluconazole
The effect of candida albicans there is no research to report at present, have wide Research Prospects.
Summary of the invention
In view of the above shortcomings of the prior art, goal of the invention of the invention is to overcome current antifungal agent resistance and hydrochloric acid to go
The larger problem of the toxic side effect of the higher generation of hydrogen banisterine usage amount provides a kind of Harmine HCl joint fluorine health
The antifungal products of azoles and its application.
To achieve the goals above, the present invention adopts the following technical scheme:
The first aspect of the invention provides a kind of Harmine HCl joint Fluconazole and is preparing anti-Candida albicans
Application in bacterium product.
Preferably, the product is drug.
Preferably, the Candida albicans is drug resistance Candida albicans.
The minimum inhibitory concentration of Harmine HCl when the use in conjunction are as follows: 8 μ g/mL.
The minimum inhibitory concentration of Fluconazole when the use in conjunction are as follows: 0.25 μ g/mL or 0.5 μ g/mL.
Preferably, the effective concentration when Harmine HCl and fluconazole are applied matches are as follows: Fluconazole:
Harmine HCl=1:32 or 1:16.
The second aspect of the invention provides a kind of antifungal medicine composition, which is with hydrochloric acid dehydrogenation
Banisterine and Fluconazole are main active.
Preferably, the effective concentration of the Harmine HCl and Fluconazole proportion is Fluconazole: hydrochloric acid dehydrogenation white horse with a black mane
Fluffy alkali=the 1:32 or 1:16 of camel.
Preferably, the minimum concentration of the Harmine HCl are as follows: 8 μ g/mL.
Preferably, the minimum concentration of the Fluconazole are as follows: 0.25 μ g/mL or 0.5 μ g/mL.
The result shows that Harmine HCl more than or equal to 8 μ g/mL be more than or equal to 0.25 μ g/mL or 0.5 μ g/
The fluconazole of mL, which is applied, has collaboration overriding resistance Candida albicans effect.
It is further preferred that the concentration of Harmine HCl and Fluconazole is respectively as follows: 8~128 μ when use in conjunction
G/mL, 0.25~64 μ g/mL.
Compared with prior art, the present invention having the following advantages that and effect:
1. present invention demonstrates that Harmine HCl and fluconazole are in application, can be enhanced Fluconazole to drug resistance
The antibacterial activity of Candida albicans, can produce collaboration antifungic action, and can reversing drug resistance Candida albicans to Fluconazole
Drug resistance, be the exploitation of new drug and old medicine newly with providing research direction.To the Candida albicans of fluconazole resistant, joint is used
Medicine can be such that its minimum inhibitory concentration is substantially reduced, and the Harmine HCl of 8 μ g/mL and 0.25 μ g/mL (read drug resistance white
Pearl bacterium CA3) or 0.5 μ g/mL (to drug resistance Candida albicans CA4) Fluconazole share can kill 80% or more fungi, it is dense
Degree increases again, and effect is stronger.
2. the pharmacological action of Harmine HCl is extensive, to the antimycotic synergistic effect of Fluconazole, it can be expanded
Application range, and minimum effective Mlc of Fluconazole is reduced, the dosage of fluconazole as antifungal medicine is reduced, to drop
The generation of the adverse reaction of low drug overcomes the problems, such as clinically antifungal agent resistance.
3. present invention demonstrates that the effect of Harmine HCl anti-candida albicans can be remarkably reinforced in Fluconazole, and dropping
The low minimum effective concentration of Harmine HCl, reduces the dosage of Harmine HCl antifungal drug,
To reduce the stronger toxic side effect of Harmine HCl.
4. antifungal agent resistance mechanism and its complexity are still still not very clear, as can by hydrochloric acid harmine there are many mechanism
Alkali is thorough to the antimycotic enhanced sensitivity Mechanism Study of Fluconazole, must can be to overcome the problems, such as that antifungal agent resistance provides new thinking.
Detailed description of the invention
It constitutes a part of Figure of description of the invention to be used to provide further understanding of the present invention, signal of the invention
Examples and descriptions thereof are used to explain the present invention for property, does not constitute improper limitations of the present invention.
Fig. 1: Harmine HCl and Fluconazole are combined to overriding resistance Candida albicans CA3 exercising result；
Fig. 2: Harmine HCl and Fluconazole are combined to overriding resistance Candida albicans CA4 exercising result；
Wherein: X-axis represents the concentration of Fluconazole, and Y-axis represents the concentration of Harmine HCl, and what Z axis represented is each
Δ E value under a pharmaceutical composition, the value higher or lower than plane (Δ E=0) respectively indicate collaboration or antagonism, around plane
Value indicate unrelated effect, in the color encoding strip for scheming right side, illustrate that synergistic effect is stronger closer to upper end.
Fig. 3: Harmine HCl and Fluconazole are combined to overriding resistance Candida albicans mycelia exercising result；
Wherein: control group (no drug, A-C), Fluconazole group (1 μ g/mL, D-F), Harmine HCl group (32 μ g/
ML, G-I), drug combination group (1 μ g/mL+ Harmine HCl of Fluconazole 32 μ g/mL, J-L).
Specific embodiment
It is noted that described further below be all exemplary, it is intended to provide further instruction to the present invention.Unless another
It indicates, all technical and scientific terms used herein has usual with general technical staff of the technical field of the invention
The identical meanings of understanding.
It should be noted that term used herein above is merely to describe specific embodiment, and be not intended to restricted root
According to exemplary embodiments of the present invention.As used herein, unless the context clearly indicates otherwise, otherwise singular
Also it is intended to include plural form, additionally, it should be understood that, when in the present specification using term "comprising" and/or " packet
Include " when, indicate existing characteristics, step, operation and/or their combination.
As background technique is introduced, long-term, high-dose is continuous using the drug resistance that Fluconazole can lead to Candida albicans
Increase, in addition, long-term, high-dose can also generate larger toxicity to body using Harmine HCl, limits hydrochloric acid dehydrogenation
The application of banisterine, in order to solve technical problem as above, the invention proposes a kind of Harmine HCls to combine fluorine
Health azoles is preparing the application in overriding resistance Candida albicans product.
Antibacterials joint can behave as four kinds of " unrelated ", " addition ", " collaboration " and " antagonism " in vitro or in animal body
Effect, for inhomogeneous drug combination, due to mechanism of action and the mode of action difference of drug, it is more likely that will increase toxicity
Or there is antagonism because of the same target position of the generation or competition that induce inactivator；Moreover, different establish a capital of same class drug has
Identical effect, such as cephalosporins, the main resisting gram-positive bacteria of generation cephalo, mainly anti-leather is blue for three, four generation cephalosporins
Family name's negative bacterium, and the third generation cephalosporin kind of only part and four generation cephalos just have the function of resisting pseudomonas aeruginosa, therefore,
For the combination of antibacterials, the antibacterial effect after being used in combination has great unpredictability.
To overcome Candida albicans to the drug resistance of Fluconazole, the present inventor has found hydrochloric acid by a large amount of experimental study
The improved effect of accident in overriding resistance Candida albicans is administered in yageine and fluconazole, when hydrochloric acid dehydrogenation white horse with a black mane
When the fluffy alkali of camel is applied alone, minimum inhibitory concentration is 64 or 128 μ g/mL, when being less than the value, very to the effect of Candida albicans
It is weak, when only be higher than 64 or 128 μ g/mL concentration under just can to Candida albicans generate bacteriostasis；And by hydrochloric acid dehydrogenation white horse with a black mane
When the fluffy alkali of camel and Fluconazole are combined, the effect of strong synergistic overriding resistance Candida albicans is showed, and being capable of reversing drug resistance white
For candida albicans to the drug resistance of Fluconazole, and when use in conjunction, the concentration of Harmine HCl is lower, reduce be applied alone compared with
The toxic side effect generated when high dose Harmine HCl.
The concentration of two kinds of drug combinations will affect the interaction of the two, in order to enable Harmine HCl and fluorine health
With certain synergistic function when azoles use in conjunction, in the preferred embodiment of the invention, the hydrochloric acid harmine
Effective concentration when alkali and fluconazole are applied matches are as follows: Fluconazole: Harmine HCl=1:32 or 1:16.
Wherein, when the application Harmine HCl minimum inhibitory concentration are as follows: 8 μ g/mL；Fluorine health when the application
The minimum inhibitory concentration of azoles are as follows: 0.25 μ g/mL or 0.5 μ g/mL.
In the preferred embodiment of the invention, when use in conjunction, selected drug concentration are as follows: gone more than or equal to 8 μ g/mL hydrochloric acid
Hydrogen banisterine be more than or equal to 0.25 μ g/mL or 0.5 μ g/mL Fluconazole.
It is further preferred that the concentration of Harmine HCl and Fluconazole is respectively as follows: 8~128 μ when use in conjunction
G/mL, 0.25~64 μ g/mL.
In the one of preferred embodiment of the present invention, a kind of antifungal medicine composition is additionally provided, the medicine group
Closing object is using Harmine HCl and Fluconazole as main active.
Wherein, the effective concentration when Harmine HCl and fluconazole are applied matches are as follows: Fluconazole: salt
Sour yageine=1:32 or 1:16；The minimum concentration of Harmine HCl when the application are as follows: 8 μ g/mL；It is described
Using when Fluconazole minimum concentration are as follows: 0.25 μ g/mL or 0.5 μ g/mL.
In another preferred embodiment of the invention, a kind of antimycotic preparation is also provided, by Harmine HCl
It is formed with Fluconazole and pharmaceutically acceptable auxiliary material.In preparation provided by the invention, pharmaceutically acceptable auxiliary material is
In pharmaceutical preparation customary adjuvant (such as: disintegrating agent, lubricant, binder etc.；Preferably one of glycerol, butanediol etc.
Or a variety of), think that feasible customary adjuvant is within the scope of the present invention for those skilled in the art, the present invention is herein
Without limitation.
It is a discovery of the invention that Harmine HCl can play in vitro with fluconazole as antifungal medicine cooperates with antimycotic work
With.
The present invention is studied using drug resistance albicans cell, using chessboard broth microdilution antifungal susceptibility test, is evaluated respectively
The joint antifungic action of different drug combination combinations.Particular content is as follows:
A: Harmine HCl and fluconazole overriding resistance Candida albicans act on: micro dilute using chessboard meat soup
Minimum effective concentration when interpretation of the law measures drug combination selects optimal drug combined concentration and evaluation combination therapies with FICI method
Effect.In addition, drug sensitive detection number will be combined for the degree that more intuitively performance drug combination acts on drug resistance Candida albicans
Further analysis has been carried out according to growth percentage difference model (Δ E).
B: Harmine HCl is found to have extensive pharmacological action, if any antibacterium, antiplasmodial, it is antimycotic,
It is anti-oxidant, antitumor etc., it can play a role in terms of overriding resistance fungal infection significant.The study show that hydrochloric acid dehydrogenation white horse with a black mane
The fluffy alkali use in conjunction Fluconazole of camel can produce collaboration overriding resistance Candida albicans effect, can expand its application range, have good
Application prospect.
In short, the present invention cooperates with overriding resistance Candida albicans using generation with fluconazole comprising Harmine HCl
The effect of bacterium.It is above-mentioned be found to be combined clinical medication as treatment drug resistance fungal infection scheme provide thinking.
Other than the activity with anti-candida albicans planktonic cells is applied alone, inventor is studying Harmine HCl
In also chance on it and can be combined to generate with Fluconazole and significantly cooperate with antifungic action.Harmine HCl and Fluconazole
Use in conjunction has collaboration overriding resistance Candida albicans effect in vitro, and effect is obvious.The hydrochloric acid harmine of low dosage
Alkali (8 μ g/mL) can make Fluconazole be down to 0.25 μ g/mL from 512 μ g/mL or more to the MIC value of drug resistance Candida albicans (to CA3)
Or 0.5 μ g/mL (to CA4).
In order to enable those skilled in the art can clearly understand technical solution of the present invention, below with reference to tool
The embodiment of the body technical solution that the present invention will be described in detail.
1 Harmine HCl of embodiment and fluconazole antifungic action measure
1. material
1.1 drugs and reagent
Fluconazole (Fluconazole, FLC), Dalian U.S. logical sequence Technology Co., Ltd.；
Harmine HCl (Ambroxol hydrochloride, HAR), the limited public affairs of Dalian U.S. logical sequence technology
Department；
Kerma (unit of kinetic energy) praises Candida chromogenic medium, Zhengzhou Bo Sai bioengineering Co., Ltd；
TTC- sand Borrow's culture medium, Qingdao high-tech park Hai Bo Bioisystech Co., Ltd；
Yeast extract, Beijing extensive and profound in meaning star biotechnology Co., Ltd；
Peptone, Beijing extensive and profound in meaning star biotechnology Co., Ltd；
Glucose, Sinopharm Chemical Reagent Co., Ltd.；
Agar powder, Beijing DingGuo ChangSheng Biology Technology Co., Ltd；
PBS phosphate buffer, Beijing DingGuo ChangSheng Biology Technology Co., Ltd；
Sodium hydroxide, state-run Shandong Dan County Organic Chemical Plant, lot number 940420；
Potassium dihydrogen phosphate, the new precious Fine Chemical Works in Shanghai, lot number 200602132.
1640 raw material medicinal powder of RPMI, GIBCO company, the U.S.；
3- (N- morpholino) propane sulfonic acid (MOPS), Beijing DingGuo ChangSheng Biology Technology Co., Ltd；
Menadione (Menadione), Sigma Co., USA；
XTT (dimethoxy azoles is yellow), Nanjing optically-active Science and Technology Ltd.；
Lactated Ringer'S Solution (ringer's solution), Shandong Lukang Cisen Pharmaceutical Co., Ltd；
Acetone, one factory of Shanghai development chemical industry, lot number 200209510；
The preparation of XTT- menadione solution: taking XTT powder 0.0500g, is dissolved in 100mL autoclaved ringer's solution
It is made into the solution of 0.5mg/mL, is gone out with 0.22 μm of filter membrane filtration；The menadione acetone soln that the 10mmol/L of 10 μ L is added (takes first
Naphthoquinones 0.0860g is dissolved in 5mL acetone), make its final concentration of 1 μm of ol/L, shakes up, 4 DEG C are kept in dark place.
Drug solution: Fluconazole is dissolved with sterile distilled water, is made into the stock solution of 2560 μ g/mL, filtering packing；Hydrochloric acid is gone
Hydrogen banisterine is dissolved with sterile distilled water, is made into the stock solution of 4096 μ g/mL, filtering packing.All medical fluids are in -20 DEG C of refrigerators
It saves, it is spare.
PBS buffer solution: 12gPBS phosphate buffer is weighed to 1L volumetric flask, distilled water stirring, which is added, keeps it completely molten
Solution, then plus distilled water to graduation mark, be dispensed into after reagent bottle the high pressure sterilization 30min at 121 DEG C, 4 DEG C of refrigerators put after cooling and are protected
It deposits.
Yeast extract-peptone-glucose agar medium: glucose 10g, peptone 10g, yeast extract 5g, agar
Powder 10g is dissolved in water in 500mL conical flask, 121 DEG C of sterilizing 30min after stirring, and 4 DEG C of refrigerators save after cooling
It is spare.
1640 liquid medium of RPMI: taking RPMI 1640 (containing L-Glutamine, being free of sodium bicarbonate) powder 2.08g,
10% glucose solution 40ml (containing sugared final concentration 2%) and MOPS powder 6.906g is added, adds distilled water to about 200mL, mixing
At 22 DEG C with the NaOH solution tune pH of 1mol/L it is about 7.0 ± 0.1 after uniformly, is filtered go out with 0.22 μm of composite fibre film before use
Bacterium.
1.2 instrument
1.3 experimental strain
Quality-control strains: Candida albicans ATCC10231 is purchased from Chinese industrial Microbiological Culture Collection administrative center；
Experimental strain: the Candida albicans that Jinan Children's Hospital is clinically separated；
Bacterial strain identification: experiment is cultivated 48 hours with bacterial strain in the case where Kerma (unit of kinetic energy) praises 35 DEG C of Candida chromogenic medium, and bacterium colony is in green
Color or emerald all bacterial strains are again through Shandong Center for Disease Control & Prevention's microbe research room in standard microbiology method
It is accredited as Candida albicans.
Bacterium solution preparation: the Candida albicans saved at -20 DEG C thaws at room temperature, is inoculated into TTC- sand Borrow's agar medium
On, 35 DEG C of cultures for 24 hours, take well-developed single bacterium colony to be inoculated with again, and 35 DEG C are cultivated for 24 hours, to guarantee that bacterial strain is in growth period.
Choose it is several individually compared with macrocolony, PBS is configured to bacteria suspension, with Chinese bacterial turbidity standard pipe ratio after vortice shaken well
Turbid, adjustment sample cell is consistent with standard pipe turbidity, and the bacteria concentration of Candida albicans is about 1 × 10 at this time6CFU/mL is serially diluted
Work bacterium solution is obtained, and concentration verifying is carried out with count plate.
2. content and method
2.1 Harmine HCls and fluconazole overriding resistance Candida albicans interaction in vitro measure
2.1.1 Harmine HCl and the effect of fluconazole overriding resistance Candida albicans measure
According to the chessboard method of CLSI M27-A3 scheme, 4 times are become with RPMI-1640 fluid nutrient medium dilute liquid medicine
Working concentration, the concentration range of screening Harmine HCl and fluconazole application, i.e. Harmine HCl are whole
Concentration is 128~2 μ g/mL, final concentration of 64~0.125 μ g/mL of Fluconazole.It is drawn respectively by the sequence of concentration from low to high
50 μ L of Fluconazole medical fluid, is separately added into the 2nd~11 column of 96 hole plates, draws hydrochloric acid dehydrogenation white horse with a black mane by the sequence of concentration from low to high
The fluffy 50 μ L of alkali medical fluid of camel, is separately added into G~A row of each 96 hole plate, and each hole adds 100 μ L bacterium solutions respectively again in addition to the 12nd column,
Remaining is supplied less than the hole of 200 μ L with RPMI-1640 culture solution.Wherein H1 is growth control, containing only bacterium solution not drug containing, the 12nd
It is classified as blank control, containing only RPMI-1640 fluid nutrient medium.According to the requirement of CLSI M27-A3 scheme, 96 holes of dosing are put down
Plate is set cultivated for 24 hours in 35 DEG C of constant incubators after, load to measure with microplate reader after 2h with XTT respectively and OD and record result.It is all
Experiment is in triplicate.
2.1.2 evaluation method and result judgement
2.1.2.1 Loewe additivity is theoretical
The basic thought of Loewe additivity (LA) theory thinks that drug can not interact with itself,
Therefore drug is applied alone or is combined and generated the concentration (equivalent site) of identical drug effect and be compared.Its analysis method score is antibacterial dense
It spends index method (fractional inhibitory concentration index, FICI), is expressed as follows:
Σ FIC=FICA+FICB=CA/MICA+CB/MICB
MICAAnd MICBIt is minimal inhibitory concentration when drug A and B are applied alone, C respectivelyAWith CBTo reach phase when the combination of two medicines
Respective concentration when with drug effect.FICI > 4 are antagonism, and FICI is added between 0.5 and 4 or unrelated effect, FICI≤
0.5 is defined as acting synergistically.
2.1.2.2 Bliss independence is theoretical
Bliss independence (BI) theory is that (fungi grows the theoretical value of percentage under each concentration of drug with Δ E
And the difference of experiment value) it is Data Analysis Model.Δ E model formulation is as follows:
Δ E=EA×EB-Ecomb
Wherein, EAFor the growth rate of A prescription used time fungi, EBFor the growth rate of B prescription used time fungi, EcombFor A medicine and B
The growth rate of fungi when medicine is combined.When interpretation of result, by Σ SYN and Σ ANT, i.e. all positive value delta E and negative value in 96 orifice plates
The interaction that the adduction of Δ E is index to judge two kinds of drugs.When the absolute value of Δ E < 100%, be expressed as weak collaboration or
Weak antagonism；When the absolute value of Δ E is between 100% and 200%, it is expressed as medium collaboration or antagonism, as Δ E
Absolute value be greater than 200% when, be expressed as Qiang Xietong or antagonism.
2.2 Harmine HCls and the effect of fluconazole overriding resistance Candida albicans mycelia measure
By this test used in the bacterial strain CA3 that is clinically separated at least passed on YPD Agr culture medium twice, use
Drug resistance Candida albicans CA3 bacterium solution is made into 1 × 10 by sterile RPMI1640 fluid nutrient medium5CFU/mL (uses Chinese bacterium
Turbidity standard pipe), drug and above-mentioned bacterium solution is added by following be grouped into the different holes of 96 orifice plates:
No. 1 hole: 200 μ L bacterium solutions；
No. 2 holes :+50 μ LRPMI1640 fluid nutrient medium of 100 4 μ g/mL Fluconazole of+50 μ L of μ L bacterium solution；
No. 3 holes :+50 μ LRPMI1640 fluid nutrient medium of+50 μ L128 μ g/mL Harmine HCl of 100 μ L bacterium solution；
No. 4 holes: 4 μ g/mL Fluconazole of+50+50 μ L of μ L128 μ g/mL Harmine HCl of 100 μ L bacterium solution.
Sterile 96 orifice plate is put and after constant temperature incubation 4h, is washed into isothermal vibration incubator (75rpm, 35 DEG C) with sterile PBS
Cell nonadherent on coverslip is removed, is put to fluorescence microscopy microscopic observation each group hole mycelia upgrowth situation.All tests repeat three
It is secondary.
2.1.3 result
2.1.3.1 minimum effective Mlc of Harmine HCl and fluconazole
The calculation method of fungi growth percentage in each hole are as follows:
Fungi grows percentage=(each hole OD value-blank control wells OD value)/growth control hole OD value
Percentage is grown by hole fungi each in above-mentioned formula calculate flat board, takes and is able to suppress the minimum of fungi growth 80%
It is interpretation terminal with drug concentration.
When Harmine HCl and fluconazole antifungic action, strong collaboration is presented to drug resistance Candida albicans and is made
With, but without synergistic effect for the sensitive strain of Candida albicans and non-white candida albicans.Now persister CA3 and CA4 is being added
Growth percentage experimental result in 96 orifice plate of medicine is listed as follows shown in (table 1-3).
Table 1. indicates that the drug combination overriding resistance Candida albicans CA3 of Harmine HCl and Fluconazole is raw with chessboard
Long percentage (sharing italic with drug the best use group that FICI method converts to mark).
Table 2. indicates that the drug combination overriding resistance Candida albicans CA4 of Harmine HCl and Fluconazole is raw with chessboard
Long percentage (sharing italic with drug the best use group that FICI method converts to mark).
2.1.3.2 it acts synergistically associated with FICI model and Δ E model evaluation Harmine HCl and Fluconazole
The evaluation index of FICI model is FICI value, and FICI≤0.5 item is defined as acting synergistically.As can be seen from Table 3, respectively
Combined FICI value is respectively less than 0.5, shows as stronger synergistic effect.
The evaluation index of Δ E model is Σ SYN and Σ ANT, as can be seen from Table 3, the summation of each group ∑ SYN and ∑ ANT
It is far longer than 200%, shows as strong synergistic effect, Harmine HCl and Fluconazole combination confrontation CA3 and CA4
Three-dimensional figure such as Fig. 1 and Fig. 2.It can intuitively find out that different pharmaceutical combination all has strong synergistic effect from figure.Picture
It is with uniformity with above-mentioned numerical analysis.
Table 3. comments acid hydrochloride salt yageine and the antimycotic work of fluconazole medication with FICI model and Δ E model
With
It explains: MIC: minimum inhibitory concentration；MICA: the minimum inhibitory concentration of Fluconazole when drug is applied alone；CA: drug combination
When Fluconazole minimum inhibitory concentration；MICB: the minimum inhibitory concentration of Harmine HCl when drug is applied alone；CB: drug connection
The minimum inhibitory concentration of used time Harmine HCl；FICI: score Mlc index；In Σ SYN:96 orifice plate it is all just
It is worth the sum of Δ E；The sum of all negative value Δ E in Σ ANT:96 orifice plate.
2.1.3.3 Harmine HCl and Fluconazole are combined the effect to mycelia
The upgrowth situation of drug resistance Candida albicans mycelia after this test is applied alone and is combined with fluorescence microscope drug.It is aobvious
It can clearly be observed that the mycelia in each group hole under micro mirror.As shown in figure 3, group and hydrochloric acid dehydrogenation is applied alone in control group, Fluconazole
Banisterine is applied alone group to show the presence of a large amount of mycelia, but finds in drug combination group: compared with other three groups, carefully
Born of the same parents' quantity significantly reduces, and generates substantially without mycelia.From this we may certainly infer that: Fluconazole and Harmine HCl are combined
It can inhibit the growth of drug resistance Candida albicans mycelia, be all substantially reduced so as to cause the drug resistance and virulence of Candida albicans.
Above-mentioned, although the foregoing specific embodiments of the present invention is described with reference to the accompanying drawings, not protects model to the present invention
The limitation enclosed, those skilled in the art should understand that, based on the technical solutions of the present invention, those skilled in the art are not
Need to make the creative labor the various modifications or changes that can be made still within protection scope of the present invention.
Claims (3)
1. a kind of Harmine HCl joint Fluconazole is preparing the application in anti-candida albicans product, characterized in that
The product is drug；
The Candida albicans is drug resistance Candida albicans；
When use in conjunction, the concentration of Harmine HCl and Fluconazole is respectively as follows: 8~128 μ g/mL, 0.25~64 μ g/
mL；
When use in conjunction, effective concentration proportion are as follows: Fluconazole: Harmine HCl=1:32 or 1:16.
2. application as described in claim 1, it is characterized in that:
The minimum inhibitory concentration of Harmine HCl when the use in conjunction are as follows: 8 μ g/mL；
The minimum inhibitory concentration of Fluconazole when the use in conjunction are as follows: 0.25 μ g/mL or 0.5 μ g/mL.
3. a kind of antifungal medicine composition, it is characterized in that: the pharmaceutical composition is with Harmine HCl and Fluconazole
For main active；
Wherein, effective concentration when Harmine HCl and fluconazole are applied matches are as follows: Fluconazole: hydrochloric acid dehydrogenation white horse with a black mane
Fluffy alkali=the 1:32 or 1:16 of camel；
The minimum concentration of Harmine HCl are as follows: 8 μ g/mL；
The minimum concentration of Fluconazole are as follows: 0.25 μ g/mL or 0.5 μ g/mL.
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| 去氢骆驼蓬碱通过下调COX-2表达抑制胃癌细胞迁移和侵袭;孙坤等;《实用老年医学》;20140228;第28卷(第2期);第117-120页 * |
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