CN108578400A - Application of the nevadensin in preparing anti-candida albicans drug - Google Patents
Application of the nevadensin in preparing anti-candida albicans drug Download PDFInfo
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- CN108578400A CN108578400A CN201810810850.6A CN201810810850A CN108578400A CN 108578400 A CN108578400 A CN 108578400A CN 201810810850 A CN201810810850 A CN 201810810850A CN 108578400 A CN108578400 A CN 108578400A
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
- A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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Abstract
The invention discloses application of the nevadensin in preparing anti-candida albicans drug.The present inventor with Candida albicans be for trying object, screening efficiently, low toxicity, be not likely to produce the compound of drug resistance, find nevadensin to the adhesiveness of Candida albicans and pathogenic there is good inhibiting effect.Moreover, compound toxicity itself is smaller, the growth of human cell is not influenced；The normal growth of Candida albicans is not influenced simultaneously, show the compound to the effect of albicans strain not mainly by kill Candida albicans, but by inhibiting the adhesiveness of Candida albicans, biofilm formation and pathogenic, therefore it is not likely to produce drug resistance.This has good application prospect in terms of the exploitation of the exploitation of novel antifungal drugs, especially anti-candida albicans infection medicine.
Description
Technical field
The invention belongs to biomedicine technical fields, more particularly to nevadensin is in preparing anti-candida albicans drug
Using.
Background technology
Candida albicans (Candida albicans) is the fungal disease of the wide-scale distribution in the mankind, is a kind of important
Opportunistic fungus, it will usually cause acute, subacute or chronic infection, and the most important disease of hospital acquired infections now
One of original.In healthy human body mucous membrane surface, such as oral cavity, enteron aisle, Candida albicans will not usually cause disease, but in immune system
It is damaged or inhibits in patient body, in chemotherapy patients, organ transplant patients or aids patient, serious system can be caused
Sexuality dye, lethality are up to 40%.
Clinically antifungal species are limited at present, and wherein azole drug (Fluconazole) is widely used, and Fluconazole is
Antibacterial effect is played by inhibiting fungi to replicate, but with the abuse of antibiotic, the phenomenon that drug resistance is increasingly severe.
Invention content
The shortcomings that it is an object of the invention to overcome the prior art and deficiency, provide nevadensin and are preparing anti-Candida albicans
Application in bacterium drug.
The purpose of the invention is achieved by the following technical solution：Nevadensin answering in preparing anti-candida albicans drug
With.
No. CAS of the nevadensin is 152743-19-6, and structural formula is as follows：
Specifically, the anti-candida albicans refer to inhibiting the adhesiveness of Candida albicans, biofilm formation, pathogenic
(to the toxicity action of Candida albicans).
The anti-candida albicans drug includes the drug for preventing and/or treating candida albicans infection, and
For preventing and treating the microbial infectious disease medicament of Candida albicans.
The invention has the advantages that：
The present invention pointedly screens efficient, low toxicity, the compound for being not likely to produce drug resistance in previous work.Then with
Candida albicans (Candida albicans) is for trying object, having investigated the nevadensin of the invention screened to Candida albicans
Adhesiveness, the influence of biofilm formation and cytotoxicity, it is therefore an objective to by detect nevadensin to Candida albicans
The interference of virulence formative factor further influences the dissemination of Candida albicans.The results show that nevadensin is to Candida albicans
The adhesiveness of bacterium, biofilm formation and pathogenic there is good inhibiting effect.Moreover, nevadensin toxicity itself is smaller, no
Influence the growth of human cell；The normal growth for not influencing Candida albicans simultaneously, shows nevadensin to Candida albicans bacterium
The effect of strain is mainly by killing Candida albicans, but by inhibiting the adhesiveness, pathogenic of Candida albicans,
And/or inhibit biofilm formation, therefore it is not likely to produce drug resistance.This is in the exploitation of novel antifungal drugs, especially anti-white
There is good application prospect in terms of the exploitation of monilial infection drug.
Therefore, nevadensin prepare anti-candida albicans infection drug in application, and prepare prevent and/or
The application in the drug of the microbial infectious diseases of Candida albicans is treated, it should all be within protection scope of the present invention.
Description of the drawings
Fig. 1 is influence result figure of the nevadensin to Candida albicans in terms of A549 cytopathics；Wherein, scheme (A)
It is cytotoxicity testing result figure of final concentration of 100 μM of the nevadensin to A549 cells；Figure (B) is that the stone of various concentration is hung
Lan Su is to the testing result figure after Candida albicans infected cell；The average result that 4 biology repeats is shown in data, accidentally
Poor stick reflects standard deviation.
Fig. 2 is influence result figure of the nevadensin to Candida albicans growth rate；Wherein, DMSO is as a contrast；Data
The average result that 3 biology repeats is shown, error bar reflects standard deviation.
Fig. 3 is influence result figure of the nevadensin to Candida albicans adhesiveness；Wherein, DMSO, BDSF be as a contrast；Number
The average result repeated according to 4 biology is shown, error bar reflect standard deviation.
Fig. 4 is influence result figure of the nevadensin to Candida albicans biofilm formation；Wherein, DMSO, BDSF conduct pair
According to；The average result that 8 biology repeats is shown in data, and error bar reflects standard deviation.
Specific implementation mode
Present invention will now be described in further detail with reference to the embodiments and the accompanying drawings, but embodiments of the present invention are unlimited
In this.
Unless stated otherwise, the present invention uses reagent, method and apparatus for the art conventional reagent, method and are set
It is standby.
Unless stated otherwise, following embodiment agents useful for same and material are purchased in market.
1 nevadensin antibacterial activity of embodiment detects
1, test method：
(1) activation of albicans strain：
Candida albicans reference culture SC5314 (being also ATCC MYA-2876) is activated into (pancreas egg in LB solid mediums
White peptone 10g/L, yeast extract 5g/L, NaCl 10g/L, agar 15g/L), it is placed in 30 DEG C of incubator overnight incubations.
(2) influence of the nevadensin to albicans strain SC5314 cytotoxicities：
(a) recovery and culture of Non-small cell lung carcinoma cell line A549 cells：The A549 cells of freeze thawing are transferred to and are contained
In the DMEM culture mediums (Gibco companies) of 10% (v/v) FBS, 37 DEG C, 5%CO2Under the conditions of be incubated overnight.
(b) A549 cells prepare：A549 cells in the high glucose medium DMEM of the fetal calf serum containing 10%vol, with 1.5 ×
104The cell concentration in a/hole overnight incubation in 96 orifice plates.When waiting for that cell is covered with 96 orifice plate bottom 80%, culture is discarded
Liquid cleans cell 3 times with 1 × PBS.
(c) Candida albicans prepares：The fresh SC5314 of picking is inoculated in the GMM culture solutions (Portugals 6.7g/L YNB, 0.2%wt
Grape sugar) in, shaken cultivation is stayed overnight under the conditions of 30 DEG C, 200rpm；It is adjusted with cell maintenance medium (DMEM of the FBS containing 1%vol)
To OD600=1.0, then dilute 10 times of (≈ 10 with cell maintenance medium8Cfu/mL), bacteria-containing cell maintenance medium is obtained.
(d) cytotoxicity bioassay：
A) nevadensin is dissolved with DMSO, prepares the nevadensin mother liquor of a concentration of 2mM.
B) toxicity action of the nevadensin to cell itself is measured：Nevadensin mother liquor is diluted to 1mM with DMSO, is added
In cell maintenance medium, final concentration of 100 μM of nevadensin, test fluid A is obtained；Meanwhile control group is set, i.e., with same volume
DMSO replace nevadensin mother liquor, obtain test fluid B.By 100 holes μ L/, test fluid A and test fluid B are separately added into preparation
In good A549 cells, it is placed in 37 DEG C, 5%CO28h is cultivated in cell incubator, often handles 4 repetitions.
C) toxicity action of nevadensin and Candida albicans to cell is measured：Nevadensin mother liquor is diluted to DMSO
A concentration of 1mM, 500 μM, 250 μM, 125 μM of dilute liquid medicine, then distinguish nevadensin mother liquor and nevadensin dilution
With bacteria-containing cell maintenance medium by volume 1:9 proportioning mixing, obtain test fluid C, final concentration of the nevadensin in test fluid C
Respectively 200 μM, 100 μM, 50 μM, 25 μM, 12.5 μM；Setting only adds DMSO (dimethyl sulfoxide (DMSO)), BDSF (cis-2- simultaneously
Dodecenoic acid, along 2- dodecenoic acids, the virtuous Chemical Industry Science Co., Ltd in Shanghai) and FLC (Fluconazole) conducts pair
According to, wherein with DMSO and bacteria-containing cell maintenance medium by volume 1:9 proportioning mixing, obtain test fluid D；It (is used with BDSF mother liquors
DMSO dissolves to obtain, a concentration of 1mM) with bacteria-containing cell maintenance medium by volume 1:9 proportioning mixing, obtain test fluid E；With fluorine
Health azoles mother liquor (dissolving to obtain with DMSO, a concentration of 1mM) and bacteria-containing cell maintenance medium by volume 1:9 proportioning mixing, obtain
Test fluid F.By 100 holes μ L/, test fluid C~F is separately added into ready A549 cells, is placed in 37 DEG C, 5%CO2Cell
8h is cultivated in incubator, often handles 4 repetitions.
D) with reference to Promega company CytoToxNonRadioactive Cytotoxicity Assay operating methods
Cell LDH activity is measured, then handles data with GraphPad Prism 6.
(3) nevadensin measures albicans strain SC5314 growth effects：
Picking bacterial strain SC5314 single bacterium colonies are inoculated in GMM culture solutions (6.7g/L YNB, 0.2%wt. glucose), 30 DEG C,
200rpm shaken cultivations are stayed overnight, and bacterium solution OD is measured600, bacterium solution is diluted to OD with GMM600=0.05.By the bacterium solution with it is a concentration of
The nevadensin liquid of 1mM by volume 9:1 mixing, is added to by the amount in 300 holes μ L/ in 100 orifice plates, each processing setting 3
A repetition, while the processing for only adding DMSO is set.It is placed in growth curve analyzer, 30 DEG C, 200rpm, is measured per 2h primary
OD600It is worth, observation experiment after 2d is as a result, GraphPad Prism 6 handle data.
(4) influence of the nevadensin to albicans strain SC5314 adhesivenesses：
(a) recovery and culture of A549 cells：The A549 cells of freeze thawing are transferred to the DMEM cultures of the FBS containing 10%vol
In base (Gibco companies), 37 DEG C, 5%CO2Under the conditions of be incubated overnight.
(b) A549 cells prepare：A549 cells in the high glucose medium DMEM of the fetal calf serum containing 10%vol, with 0.5 ×
103The cell concentration in a/hole overnight incubation in 96 orifice plates.When waiting for that cell is covered with 96 orifice plate bottom 80%, culture is discarded
Liquid cleans cell 3 times with 1 × PBS.
(c) the SC5314 bacterial strains on picking LB solid plates are inoculated in the GMM culture solutions (Portugals 6.7g/L YNB, 0.2%wt
Grape sugar) in, 30 DEG C, 200rpm shaken cultivations are stayed overnight, and measure bacterium solution OD600.Then with cell maintenance medium (FBS's containing 1%vol
DMEM it) adjusts and bacterium solution is diluted to OD600=0.5.Nevadensin mother liquor is diluted to a concentration of 1mM, 500 μM, 250 μ with DMSO
Then nevadensin mother liquor, nevadensin dilution are pressed body by M, 125 μM of dilute liquid medicine with bacteria-containing cell maintenance medium respectively
Product ratio 1:9 proportioning mixing, shake mixing, obtain test fluid G, final concentration of the nevadensin in test fluid G be respectively 200 μM,
100μM、50μM、25μM、12.5μM.It is added by 100 holes μ L/ in 96 orifice plates that step (b) has cultivated cell, each processing setting
4 repetitions；Setting simultaneously is only plus a processing of DMSO and BDSF, wherein with DMSO and bacteria-containing cell maintenance medium by volume 1:9
Proportioning mixing, obtains test fluid H；It is pressed with bacteria-containing cell maintenance medium with BDSF mother liquors (dissolving to obtain with DMSO, a concentration of 1mM)
Volume ratio 1:9 proportioning mixing, obtain test fluid I.96 orifice plates are statically placed in 37 DEG C and are incubated, culture solution is discarded after 1.5h, per hole
The crystal violet solution of 100 μ L a concentration of 0.1% (w/v) is added, room temperature acts on 45min.Crystal violet is discarded, ice ddH is used in combination2O
It washes 10 times, the ethanol solution of a concentration of percents by volume of 100 μ L 75% is added, is placed at room temperature for 30 minutes, measure OD590, use
6 software data processings of GraphPad Prism.
(5) influence of the nevadensin to albicans strain SC5314 biofilm formations：
SC5314 bacterial strains on picking LB solid plates are inoculated in SDA culture solutions (40g maltose, 10g peptones, distillation
Water is settled to 1L, adjusts pH to 6.0 ± 0.2), 30 DEG C, 200rpm shaken cultivations stay overnight, measure bacterium solution OD600.Then cultivated with SDA
Bacterium solution is diluted to OD by liquid600=0.1, obtain bacteria-containing SDA culture solutions.Nevadensin mother liquor is diluted to DMSO a concentration of
1mM, 500 μM, 250 μM, 125 μM of dilute liquid medicine, then by nevadensin mother liquor, nevadensin dilution respectively with it is bacteria-containing
SDA culture solutions by volume 1:9 proportioning mixing, shake mixing, obtain test fluid J, final concentration of the nevadensin in test fluid J
Respectively 200 μM, 100 μM, 50 μM, 25 μM, 12.5 μM.It is added in 96 orifice plates by 100 holes μ L/, 8 weights of each processing setting
It is multiple, while the processing for only adding DMSO and only adding BDSF (final concentration of 100 μM of BDSF) is set.96 orifice plates are statically placed in 37 DEG C
Middle incubation discards culture solution after 8h, and 100 μ L, 0.1% crystal violets are added, and room temperature acts on 45min.Crystal violet is discarded, ice is used in combination
ddH2O is washed 10 times, and 100 μ L, 75% ethyl alcohol is added, is placed at room temperature for 30 minutes, measures OD590, with GraphPad Prism6 softwares
Handle data.
2, experimental result
(1) nevadensin has certain inhibiting effect to the virulence of albicans strain SC5314
We detect the toxicity of cell by detecting the burst size of LDH, when detecting the cytotoxicity of Candida albicans,
We will add the LDH burst sizes of DMSO groups as 100%, and thus carrys out specification other LDH that nevadensin groups are added and release
Put ratio.The results are shown in Figure 1, and the average result that 4 biology repeats is shown in data, and error bar reflects standard deviation.
Cytotoxicity experimental result is shown, is control with DMSO, under conditions of no Candida albicans, nevadensin pair
Cell does not have toxicity, as shown in Fig. 1 (A).
And under conditions of adding Candida albicans SC5314, it is the positive with DMSO, BDSF is negative control, Fig. 1 (B) displays
Nevadensin has certain protective role in inhibition bacterial strain SC5314 to infecting for cell；Nevadensin is in 100 μM of concentration, in vain
The virulence of color candida albicans is reduced to 44.2%.
(2) nevadensin does not influence the growth of albicans strain SC5314
The results are shown in Figure 2, is control with DMSO, to albicans strain at a concentration of 100 μM of nevadensin
The growth of SC5314 does not influence substantially.This is not the result shows that nevadensin is to the effect of albicans strain SC5314
Bacterium is killed, therefore is not likely to produce drug resistance.
(3) nevadensin inhibits the adhesiveness of albicans strain SC5314
As shown in figure 3, using DMSO, BDSF as reference, through final concentration of 100 μM of nevadensin treated Candida albicans
Adhesiveness of the bacterium on polystyrene, reduces 46% or so.Show that nevadensin is shown to Candida albicans SC5314
Adhesiveness have certain inhibiting effect.
(4) influence of the nevadensin to albicans strain SC5314 biofilm formations：
As shown in figure 4, using DMSO, BDSF as reference, through nevadensin (100 μM) treated Candida albicans in polyphenyl
Biofilm formation on ethylene, reduces 46.6% or so.As it can be seen that nevadensin is shown to Candida albicans SC5314's
Biofilm formation has good inhibiting effect.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, it is other it is any without departing from the spirit and principles of the present invention made by changes, modifications, substitutions, combinations, simplifications,
Equivalent substitute mode is should be, is included within the scope of the present invention.
Claims (3)
1. application of the nevadensin in preparing anti-candida albicans drug.
2. application of the nevadensin according to claim 1 in preparing anti-candida albicans drug, it is characterised in that：Institute
The anti-candida albicans drug stated is that have the adhesiveness for inhibiting Candida albicans, biofilm formation, pathogenic drug.
3. application of the nevadensin according to claim 1 in preparing anti-candida albicans drug, it is characterised in that：Institute
The anti-candida albicans drug stated is the drug for preventing and/or treating candida albicans infection, and for preventing and controlling
Treat one or both of microbial infectious disease medicament of Candida albicans.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117045641A (en) * | 2023-10-11 | 2023-11-14 | 广东医科大学附属医院 | Application of stone-like chlorophyllin in preparing toxoplasma resistant medicine |
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2018
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117045641A (en) * | 2023-10-11 | 2023-11-14 | 广东医科大学附属医院 | Application of stone-like chlorophyllin in preparing toxoplasma resistant medicine |
| CN117045641B (en) * | 2023-10-11 | 2024-01-23 | 广东医科大学附属医院 | Application of stone-like chlorophyllin in preparing toxoplasma resistant medicine |
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