CN108498784A - Application of the Radix pseudostellariae cyclic peptides B in preparing anti-candida albicans drug - Google Patents
Application of the Radix pseudostellariae cyclic peptides B in preparing anti-candida albicans drug Download PDFInfo
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- CN108498784A CN108498784A CN201810536760.2A CN201810536760A CN108498784A CN 108498784 A CN108498784 A CN 108498784A CN 201810536760 A CN201810536760 A CN 201810536760A CN 108498784 A CN108498784 A CN 108498784A
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- A61K38/00—Medicinal preparations containing peptides
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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Abstract
The invention discloses application of the Radix pseudostellariae cyclic peptides B 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 Radix pseudostellariae cyclic peptides B to the adhesiveness of Candida albicans and pathogenic there is good inhibiting effect.Moreover, Radix pseudostellariae cyclic peptides B toxicity itself is smaller, the growth of human cell is not influenced；It does not influence the normal growth of Candida albicans simultaneously, shows that Radix pseudostellariae cyclic peptides B, mainly by killing Candida albicans, but by inhibiting the adhesiveness, pathogenic of Candida albicans, therefore is not likely to produce drug resistance to the effect of albicans strain.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 field, more particularly to Radix pseudostellariae cyclic peptides B is preparing anti-candida albicans drug
In application.
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 Radix pseudostellariae cyclic peptides B and are preparing anti-white
Application in candida albicans drug.
The purpose of the invention is achieved by the following technical solution：Radix pseudostellariae cyclic peptides B is in preparing anti-candida albicans drug
Application.
No. CAS of the Radix pseudostellariae cyclic peptides B is 145459-19-4, and structural formula is as follows：
Specifically, the anti-candida albicans refer to inhibiting the adhesiveness of Candida albicans and pathogenic (to Candida albicans
The toxicity action of bacterium).
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 Radix pseudostellariae cyclic peptides B of the invention screened and read white
The adhesiveness of pearl bacterium and the influence of cytotoxicity, it is therefore an objective to by detecting Radix pseudostellariae cyclic peptides B to Candida albicans virulence
The interference of formative factor further influences the dissemination of Candida albicans.The results show that Radix pseudostellariae cyclic peptides B is to Candida albicans
The adhesiveness of bacterium and it is pathogenic have good inhibiting effect.Moreover, Radix pseudostellariae cyclic peptides B toxicity itself is smaller, the mankind are not influenced
The growth of cell；The normal growth for not influencing Candida albicans simultaneously, shows work of the Radix pseudostellariae cyclic peptides B to albicans strain
With mainly by killing Candida albicans, but by inhibiting the adhesiveness, pathogenic of Candida albicans, therefore it is not easy
Generate drug resistance.This has in terms of the exploitation of the exploitation of novel antifungal drugs, especially anti-candida albicans infection medicine
Good application prospect.
Therefore, application of the Radix pseudostellariae cyclic peptides B in the drug for preparing anti-candida albicans infection, and prevent preparing
And/or the application in the drug of the treatment microbial infectious diseases of Candida albicans, it should all be within protection scope of the present invention.
Description of the drawings
Fig. 1 is influence result figure of the Radix pseudostellariae cyclic peptides B to Candida albicans in terms of A549 cytopathics；Wherein, scheme
(A) it is testing result figure of final concentration of 100 μM of the Radix pseudostellariae cyclic peptides B to the cytotoxicity of A549 cells；Figure (B) is different dense
The Radix pseudostellariae cyclic peptides B of degree is to the testing result figure after Candida albicans infected cell.
Fig. 2 is influence result figure of the Radix pseudostellariae cyclic peptides B to Candida albicans growth rate；Wherein, DMSO is as a contrast；Number
The average result repeated according to 3 biology is shown, error bar reflect standard deviation.
Fig. 3 is influence result figure of the Radix pseudostellariae cyclic peptides B to Candida albicans adhesiveness；Wherein, DMSO, BDSF conduct pair
According to；The average result that 4 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 Radix pseudostellariae cyclic peptides B antibacterial activity of embodiment detects
1, test method：
(1) activation of albicans strain：
Candida albicans reference culture SC5314 is activated into (tryptone 10g/L, yeast extract in LB solid mediums
5g/L, NaCl 10g/L, agar 15g/L), it is placed in 30 DEG C of incubator overnight incubations.
(2) influence of the Radix pseudostellariae cyclic peptides B 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 are in the high glucose medium DMEM containing 10% fetal calf serum, with 1.5 × 104
The 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 solution is discarded,
Cell is cleaned with 1 × PBS 3 times.
(c) Candida albicans prepares：The fresh SC5314 of picking is inoculated in GMM culture solutions (6.7g/L YNB, 0.2% grape
Sugar) in, shaken cultivation is stayed overnight under the conditions of 30 DEG C, 200rpm；It is adjusted to OD with cell maintenance medium (DMEM containing 1%FBS)600
=1.0, then dilute 10 times of (≈ 10 with cell maintenance medium8Cfu/mL), bacteria-containing cell maintenance medium is obtained.
(d) cytotoxicity bioassay：
A) Radix pseudostellariae cyclic peptides B is dissolved with DMSO, prepares the Radix pseudostellariae cyclic peptides B mother liquor of a concentration of 2mM.
B) toxicity action of the Radix pseudostellariae cyclic peptides B to cell itself is measured：Radix pseudostellariae cyclic peptides B mother liquor is diluted to DMSO
1mM is added in cell maintenance medium, final concentration of 100 μM of Radix pseudostellariae cyclic peptides B, obtains test fluid A；Meanwhile control group is set,
Radix pseudostellariae cyclic peptides B mother liquor is replaced with the DMSO of same volume, obtains test fluid B.By 100 holes μ L/, by test fluid A and test
Liquid B is separately added into ready A549 cells, is placed in 37 DEG C, 5%CO28h is cultivated in cell incubator, often handles 4 weights
It is multiple.
C) toxicity action of Radix pseudostellariae cyclic peptides B and Candida albicans to cell is measured：By Radix pseudostellariae cyclic peptides B mother liquor DMSO
It is diluted to a concentration of 1mM, 500 μM, 250 μM, 125 μM of dilute liquid medicine, then by Radix pseudostellariae cyclic peptides B mother liquor and Radix pseudostellariae cyclic peptides
B dilutions respectively with bacteria-containing cell maintenance medium by volume 1:9 proportioning mixing, obtain test fluid C, Radix pseudostellariae cyclic peptides B is being surveyed
Final concentration in test solution C is respectively 200 μM, 100 μM, 50 μM, 25 μM, 12.5 μM, and in test fluid C, the volume content of DMSO is
It is identical；Setting simultaneously only adds DMSO (dimethyl sulfoxide (DMSO)), BDSF (cis-2-dodecenoic acid, along 2- dodecylenes
Acid, the virtuous Chemical Industry Science Co., Ltd in Shanghai) and FLC (Fluconazole) is as a contrast, wherein it is maintained with DMSO and bacteria-containing cell
Liquid by volume 1:9 proportioning mixing, obtain test fluid D；With BDSF mother liquors (dissolving to obtain with DMSO, a concentration of 1mM) and contain bacterium
Cell maintenance medium by volume 1:9 proportioning mixing, obtain test fluid E；It (dissolves to obtain with DMSO, concentration with Fluconazole mother liquor
For 1mM) with bacteria-containing cell maintenance medium by volume 1:9 proportioning mixing, obtain test fluid F.By 100 holes μ L/, by test fluid C
~F is separately added into ready A549 cells, is placed in 37 DEG C, 5%CO28h is cultivated in cell incubator, often handles 4 weights
It is multiple.
D) with reference to Promega company CytoToxThe operation sides NonRadioactive Cytotoxicity Assay
Method measures cell LDH activity, then handles data with GraphPad Prism 6.
(3) Radix pseudostellariae cyclic peptides B 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% 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 Radix pseudostellariae cyclic peptides B liquid of 1mM by volume 9:1 mixing, is added to by the amount in 300 holes μ L/ in 100 orifice plates, and each processing is set
3 repetitions are set, while the processing for only adding DMSO is set.It is placed in growth curve analyzer, 30 DEG C, 200rpm, one is measured per 2h
Secondary OD600It is worth, observation experiment after 2d is as a result, GraphPad Prism 6 handle data.
(4) influence of the Radix pseudostellariae cyclic peptides B to albicans strain SC5314 adhesivenesses：
(a) recovery and culture of A549 cells：The A549 cells of freeze thawing are transferred to the DMEM culture mediums containing 10%FBS
In (Gibco companies), 37 DEG C, 5%CO2Under the conditions of be incubated overnight.
(b) A549 cells prepare：A549 cells are in the high glucose medium DMEM containing 10% fetal calf serum, with 0.5 × 103
The 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 solution is discarded,
Cell is cleaned with 1 × PBS 3 times.
(c) the SC5314 bacterial strains on picking LB solid plates are inoculated in GMM culture solutions (6.7g/L YNB, 0.2% grape
Sugar) in, 30 DEG C, 200rpm shaken cultivations are stayed overnight, and measure bacterium solution OD600.Then adjusted with cell maintenance medium (DMEM containing 1%FBS)
Bacterium solution is diluted to OD by section600=0.5.By Radix pseudostellariae cyclic peptides B mother liquor with DMSO be diluted to a concentration of 1mM, 500 μM, 250 μM,
Then 125 μM of dilute liquid medicine maintains Radix pseudostellariae cyclic peptides B mother liquor and Radix pseudostellariae cyclic peptides B dilution with bacteria-containing cell respectively
Liquid by volume 1:9 proportioning mixing, shake mixing, obtain test fluid G, final concentration difference of the Radix pseudostellariae cyclic peptides B in test fluid G
It is 200 μM, 100 μM, 50 μM, 25 μM, 12.5 μM.It is added by 100 holes μ L/ in 96 orifice plates that step (b) has cultivated cell, each
Processing 4 repetitions of setting；Setting simultaneously only adds the processing of DMSO and BDSF, wherein is pressed with DMSO and bacteria-containing cell maintenance medium
Volume ratio 1:9 proportioning mixing, obtain test fluid H；With BDSF mother liquors (dissolving to obtain with DMSO, a concentration of 1mM) with it is bacteria-containing thin
Born of the same parents' maintaining liquid by volume 1:9 proportioning mixing, obtain test fluid I.96 orifice plates are statically placed in 37 DEG C and are incubated, training is discarded after 1.5h
Nutrient solution, the crystal violet solution of 100 μ L a concentration of 0.1% (w/v) is added per hole, and room temperature acts on 45min.Crystal violet is discarded, and
With ice ddH2O is washed 10 times, and 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, is measured
OD590, with 6 software data processings of GraphPad Prism.
2, experimental result
(1) Radix pseudostellariae cyclic peptides B 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 the LDH of Radix pseudostellariae cyclic peptides B groups is added in other
Releasing 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
Difference.
Cytotoxicity experimental result is shown, is compareed with DMSO, under conditions of no Candida albicans, Radix pseudostellariae cyclic peptides B
There is no toxicity to cell, 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
Radix pseudostellariae cyclic peptides B has certain protective role in inhibition bacterial strain SC5314 to infecting for cell, and in 100 μM of concentration, white is read
The virulence of pearl bacterium is reduced to 22.6%.
(2) Radix pseudostellariae cyclic peptides B 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 Radix pseudostellariae cyclic peptides B
The growth of SC5314 does not influence (two growth curves essentially coincide together).It is somebody's turn to do the result shows that Radix pseudostellariae cyclic peptides B is to white
The effect of beads bacteria strain SC5314 is not to kill bacterium, therefore be not likely to produce drug resistance.
(3) Radix pseudostellariae cyclic peptides B inhibits the adhesiveness of albicans strain SC5314
The results are shown in Figure 3, using DMSO as reference group, using the adhesiveness of DMSO reference groups as 100%, BDSF processing groups
Candida albicans adherence reduce 55%, through final concentration of 100 μM of Radix pseudostellariae cyclic peptides B, treated that Candida albicans exists
Adhesiveness on polystyrene, reduces 12.8% or so.Show that Radix pseudostellariae cyclic peptides B is shown to Candida albicans SC5314
Adhesiveness have certain 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 Radix pseudostellariae cyclic peptides B in preparing anti-candida albicans drug.
2. application of the Radix pseudostellariae cyclic peptides B according to claim 1 in preparing anti-candida albicans drug, feature exist
In：The anti-candida albicans drug is with the adhesiveness and pathogenic drug for inhibiting Candida albicans.
3. application of the Radix pseudostellariae cyclic peptides B according to claim 1 in preparing anti-candida albicans drug, feature exist
In：The anti-candida albicans drug is the drug for preventing and/or treating candida albicans infection, and for preventing
With treatment one or both of microbial infectious disease medicament of Candida albicans.
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Cited By (2)
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| CN110237232A (en) * | 2019-07-30 | 2019-09-17 | 广东海洋大学 | Radix pseudostellariae cyclic peptides B is improving the application in memory and/or senile dementia prevention and cure |
| CN113456641A (en) * | 2021-07-08 | 2021-10-01 | 中国科学院微生物研究所 | Application of compound in preparation of antifungal medicine |
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Application publication date: 20180907 |