CN108578400B - Application of lysionotin in preparation of anti-candida albicans drugs - Google Patents
Application of lysionotin in preparation of anti-candida albicans drugs Download PDFInfo
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- CN108578400B CN108578400B CN201810810850.6A CN201810810850A CN108578400B CN 108578400 B CN108578400 B CN 108578400B CN 201810810850 A CN201810810850 A CN 201810810850A CN 108578400 B CN108578400 B CN 108578400B
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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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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/10—Antimycotics
Abstract
The invention discloses application of lysionotin in preparation of anti-candida albicans drugs. The inventor selects a compound which is efficient, low-toxicity and difficult to generate drug resistance by taking candida albicans as a test object, and finds that the lysionotin has a good inhibition effect on the adhesion and pathogenicity of the candida albicans. Moreover, the compound has low toxicity and does not influence the growth of human cells; meanwhile, the compound does not influence the normal growth of the candida albicans, and shows that the effect of the compound on the candida albicans strain is not mainly based on killing the candida albicans, but is achieved by inhibiting the adhesion, biofilm formation and pathogenicity of the candida albicans, so that the compound is not easy to generate drug resistance. The compound has good application prospect in the development of novel antifungal drugs, especially in the development of drugs for resisting Candida albicans infection.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to application of lysionotin in preparation of anti-candida albicans drugs.
Background
Candida albicans (Candida albicans) is a widely spread fungal disease in humans, is an important conditionally pathogenic fungus, usually causes acute, subacute or chronic infections, and is one of the most important pathogens of hospital-acquired infections at present. Candida albicans does not normally cause disease on mucosal surfaces of healthy persons, such as the oral cavity and intestinal tract, but causes serious systemic infection in patients with compromised or suppressed immune systems, such as chemotherapy patients, organ transplant patients or AIDS patients, with mortality rates as high as 40%.
At present, clinically, antifungal medicines are limited in species, wherein azole medicines (fluconazole) are widely applied, the fluconazole plays a role in inhibiting bacteria by inhibiting fungal replication, but the phenomenon of drug resistance is more serious along with abuse of antibiotics.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide the application of the lysionotin in preparing the anti-candida albicans drug.
The purpose of the invention is realized by the following technical scheme: application of lysionotin in preparing anti-Candida albicans medicine is provided.
The CAS number of the lysionotin is 152743-19-6, and the structural formula is shown as follows:
specifically, the anti-candida albicans refers to the inhibition of the adhesion, biofilm formation and pathogenicity (the virulence effect on candida albicans) of the candida albicans.
The anti-candida albicans medicine comprises a medicine for preventing and/or treating candida albicans infection and a medicine for preventing and treating infectious diseases caused by candida albicans.
The invention has the following beneficial effects:
the invention aims to screen compounds with high efficiency, low toxicity and difficult drug resistance in earlier work. Then, Candida albicans (Candida albicans) are taken as test objects, the influence of the gelidium amansii screened by the invention on the adhesion, biofilm formation and cytotoxicity of the Candida albicans is examined, and the purpose is to further influence the infection effect of the Candida albicans by detecting the interference of the gelidium amansii on the virulence forming factors of the Candida albicans. The results show that the lysionotin has good inhibition effect on the adhesion, biofilm formation and pathogenicity of candida albicans. Moreover, the lysionotin has low toxicity and does not influence the growth of human cells; meanwhile, the normal growth of the candida albicans is not influenced, and the result shows that the effect of the lysionotin on the candida albicans strain is not mainly based on killing the candida albicans, but is achieved by inhibiting the adhesion and pathogenicity of the candida albicans and/or inhibiting the formation of a biofilm, so that the drug resistance is not easy to generate. The compound has good application prospect in the development of novel antifungal drugs, especially in the development of drugs for resisting Candida albicans infection.
Therefore, the application of the lysionotin in preparing the medicines for resisting candida albicans infection and preventing and/or treating infectious diseases caused by candida albicans should be within the protection scope of the invention.
Drawings
FIG. 1 is a graph showing the effect of Shibatalin on the pathogenicity of A549 cells of Candida albicans; wherein, the graph (A) is a graph of the cytotoxicity detection result of the shikonin with the final concentration of 100 mu M on the A549 cells; the graph (B) is a graph of the detection results of different concentrations of the lysionotin after infecting the cells with Candida albicans; data shown are the average of 4 biological replicates, and error bars reflect standard deviations.
FIG. 2 is a graph showing the effect of nevadensin on the growth rate of Candida albicans; DMSO as control, among others; data shown are the average of 3 biological replicates, and error bars reflect standard deviations.
FIG. 3 is a graph showing the effect of gelsolin on Candida albicans adhesion; wherein DMSO, BDSF are used as control; data shown are the average of 4 biological replicates, and error bars reflect standard deviations.
FIG. 4 is a graph showing the effect of nevadensin on Candida albicans biofilm formation; wherein DMSO, BDSF are used as control; data are shown as the average of 8 biological replicates, with error bars reflecting standard deviations.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
Example 1 detection of antibacterial Activity of Lysionotinum
1. The test method comprises the following steps:
(1) activation of candida albicans strains:
candida albicans standard strain SC5314 (also ATCC MYA-2876) was activated in LB solid medium (tryptone 10g/L, yeast extract 5g/L, NaCl 10g/L, agar 15g/L), and cultured overnight in a 30 ℃ incubator.
(2) Effect of nevadensin on the cytotoxicity of candida albicans strain SC 5314:
(a) and (3) recovering and culturing the human non-small cell lung cancer cell line A549 cells: freeze-thawed A549 cells were transferred to DMEM medium (Gibco Co.) containing 10% (v/v) FBS at 37 ℃ with 5% CO2Culturing overnight under the condition。
(b) Preparation of a549 cells: a549 cells in DMEM (high glucose Medium) containing 10% vol fetal bovine serum at 1.5X 104Cell concentration per well was cultured overnight in 96-well plates. When the cells were 80% full of the bottom of the 96-well plate, the culture medium was discarded, and the cells were washed 3 times with 1 × PBS.
(c) Preparation of candida albicans: selecting fresh SC5314, inoculating into GMM culture solution (6.7g/L YNB, 0.2% wt glucose), and shake culturing at 30 deg.C and 200rpm overnight; regulation to OD with cell maintenance solution (DMEM containing 1% vol FBS)6001.0, diluted 10-fold with cell maintenance medium (≈ 10)8cfu/mL) to obtain a cell maintenance solution containing the bacteria.
(d) And (3) determining the cytotoxicity:
a) lysionotinum was dissolved in DMSO to prepare a mother solution of Lysionotinum at a concentration of 2 mM.
b) Determining the toxicity effect of the lysionotin on cells: diluting the lysionotin mother liquor to 1mM with DMSO, and adding into a cell maintenance solution with a final concentration of 100 μ M to obtain a test solution A; meanwhile, a control group is set, namely DMSO with the same volume is used for replacing the lysionotin mother solution, so as to obtain a test solution B. Adding test solution A and test solution B into prepared A549 cells at 100 μ L/well, respectively, standing at 37 deg.C and 5% CO2The cells were incubated in an incubator for 8h, 4 replicates per treatment.
c) Determining the toxicity of the lysionotin and candida albicans on cells: diluting the lysionotin mother liquor with DMSO to obtain diluted liquor with concentration of 1mM, 500. mu.M, 250. mu.M and 125. mu.M, and mixing the lysionotin mother liquor and the lysionotin diluted liquor with the cell maintenance liquid containing bacteria at a volume ratio of 1:9 to obtain a test solution C, wherein the final concentrations of lysionotin in the test solution C are 200. mu.M, 100. mu.M, 50. mu.M, 25. mu.M and 12.5. mu.M respectively; simultaneously, only adding DMSO (dimethyl sulfoxide), BDSF (cis-2-dodecanoic acid, cis-2-dodecenoic acid, Shanghai has Ded chemical engineering science and technology Co., Ltd.) and FLC (fluconazole) as a control, wherein DMSO and the cell maintenance solution containing bacteria are mixed according to the volume ratio of 1:9 to obtain a test solution D; BDSF stock solution (dissolved in DMSO and having a concentration of 1mM) and cell maintenance solution containing bacteria are mixed in a liquidMixing the components according to the volume ratio of 1:9 to obtain a test solution E; and mixing the fluconazole mother solution (dissolved by DMSO and with the concentration of 1mM) and the bacteria-containing cell maintenance solution according to the volume ratio of 1:9 to obtain a test solution F. Adding the test solutions C-F into the prepared A549 cells at 100 μ L/well, respectively, and standing at 37 deg.C and 5% CO2The cells were incubated in an incubator for 8h, 4 replicates per treatment.
d) Reference is made to Promega corporation CytoTox
Cellular LDH activity was determined by the NonRadioactive cytoxicity Assay protocol followed by GraphPad Prism6 treatment of the data.
(3) Determination of the effect of geldanamycin on the growth of candida albicans strain SC 5314:
selecting single colony of strain SC5314, inoculating to GMM culture solution (6.7g/L YNB, 0.2% wt. glucose), shaking and culturing at 30 deg.C and 200rpm overnight, and determining OD of bacterial solution600Diluting the bacterial liquid to OD with GMM6000.05. The bacterial liquid and 1mM of lysionotin liquor are mixed according to the volume ratio of 9:1, and added into a 100-well plate according to the amount of 300 mu L/well, each treatment is set to be 3 times, and the treatment only adding DMSO is set at the same time. Placing in a growth curve tester, measuring OD every 2h at 30 deg.C and 200rpm600Values, observed after 2d experimental results, GraphPad Prism6 processed data.
(4) Effect of lysionotin on adhesion of candida albicans strain SC 5314:
(a) recovery and culture of A549 cells: freeze-thawed A549 cells were transferred to DMEM medium (Gibco Co.) containing 10% vol FBS at 37 ℃ with 5% CO2Cultured overnight under the conditions.
(b) Preparation of a549 cells: a549 cells in DMEM (high glucose Medium) containing 10% vol fetal bovine serum at 0.5X 103Cell concentration per well was cultured overnight in 96-well plates. When the cells were 80% full of the bottom of the 96-well plate, the culture medium was discarded, and the cells were washed 3 times with 1 × PBS.
(c) SC5314 strain on LB solid plate was picked, inoculated into GMM culture medium (6.7g/L YNB, 0.2% wt glucose), and shake-cultured at 30 ℃ and 200rpmCulturing overnight, and determining bacterial liquid OD600. The cell culture broth (DMEM containing 1% vol FBS) was then used to adjust the dilution to OD6000.5. Diluting the lysionotin mother liquor with DMSO to obtain diluted liquor with concentrations of 1mM, 500. mu.M, 250. mu.M and 125. mu.M, mixing the lysionotin mother liquor and the lysionotin diluted liquor with the cell maintenance liquid containing bacteria according to a volume ratio of 1:9, shaking and mixing uniformly to obtain a test solution G, wherein the final concentrations of lysionotin in the test solution G are 200. mu.M, 100. mu.M, 50. mu.M, 25. mu.M and 12.5. mu.M respectively. Adding 100 μ L/well into 96-well plates of cultured cells of step (b), and setting 4 repetitions for each treatment; simultaneously setting treatment of only adding DMSO and BDSF, wherein DMSO and the cell maintenance liquid containing bacteria are mixed according to the volume ratio of 1:9 to obtain a test liquid H; BDSF mother liquor (obtained by dissolving with DMSO and with the concentration of 1mM) and the cell maintenance liquid containing the bacteria are mixed according to the volume ratio of 1:9 to obtain a test liquid I. Incubating 96-well plate at 37 deg.C, discarding culture solution after 1.5 hr, adding 100 μ L crystal violet solution with concentration of 0.1% (w/v) into each well, and allowing to act at room temperature for 45 min. Discarding crystal violet and using ice ddH2O washing 10 times, adding 100 μ L ethanol solution with volume percent of 75%, standing at room temperature for 30 min, and determining OD590Data were processed using GraphPad Prism6 software.
(5) Effect of lysionotin on biofilm formation by candida albicans strain SC 5314:
selecting SC5314 strain on LB solid plate, inoculating in SDA culture solution (40g maltose, 10g peptone, distilled water to constant volume of 1L, adjusting pH to 6.0 + -0.2), shaking and culturing at 30 deg.C and 200rpm overnight, and determining OD of bacterial solution600. Then diluting the bacterial liquid to OD by using SDA culture solution600When the concentration was 0.1, a culture solution of SDA containing bacteria was obtained. Diluting the lysionotin mother liquor with DMSO to obtain diluted liquor with concentrations of 1mM, 500. mu.M, 250. mu.M and 125. mu.M, mixing the lysionotin mother liquor and the lysionotin diluted liquor with bacteria-containing SDA culture solution according to a volume ratio of 1:9, shaking and mixing uniformly to obtain test solution J, wherein the final concentrations of lysionotin in the test solution J are 200. mu.M, 100. mu.M, 50. mu.M, 25. mu.M and 12.5. mu.M respectively. Add 100. mu.L/well to 96-well plates, set up 8 replicates per treatment, set up DMSO only and BDSF only (of BDSF)Final concentration of 100. mu.M). Incubating 96-well plate at 37 deg.C, discarding culture solution after 8 hr, adding 100 μ L of 0.1% crystal violet, and allowing to act at room temperature for 45 min. Discarding crystal violet and using ice ddH2O washing 10 times, adding 100 μ L75% ethanol, standing at room temperature for 30 min, and measuring OD590Data was processed using GraphPad Prism6 software.
2. Results of the experiment
(1) The lysionotin has certain inhibitory effect on the toxicity of Candida albicans strain SC5314
The release amount of LDH is detected to detect the toxicity of cells, and when the cytotoxicity of candida albicans is detected, the release amount of LDH of a DMSO group is taken as 100%, so that the LDH release ratio of other added lysionotin groups is regulated. Results are shown in figure 1, data show the average of 4 biological replicates, and error bars reflect standard deviations.
The results of the cytotoxicity experiments show that the nevadensin has no toxicity to the cells under the condition without candida albicans by taking DMSO as a control, as shown in figure 1 (A).
Under the condition of adding candida albicans SC5314, DMSO is used as positive, BDSF is used as negative control, and figure 1(B) shows that the lysionotin has a certain protection effect on the infection of cells by inhibiting the strain SC 5314; the virulence of Candida albicans was reduced to 44.2% at a concentration of 100. mu.M lysin.
(2) The lysionotin has no effect on the growth of Candida albicans strain SC5314
The results are shown in FIG. 2, where the concentration of lysionotin was 100. mu.M and the control was DMSO, which had substantially no effect on the growth of C.albicans strain SC 5314. The results indicate that the effect of nevadensin on candida albicans strain SC5314 is not a bacterial kill and therefore is not susceptible to drug resistance.
(3) Adherence of geldanamycin to Candida albicans strain SC5314
As shown in FIG. 3, the adhesion of Candida albicans treated with nevadensin at a final concentration of 100 μ M to polystyrene was reduced by about 46% with DMSO and BDSF as reference. The results show that the lysionotin has a certain inhibition effect on the adhesion of candida albicans SC 5314.
(4) Effect of lysionotin on biofilm formation by candida albicans strain SC 5314:
as shown in FIG. 4, the biofilm formation of Candida albicans on polystyrene was reduced by about 46.6% after treatment with lysionotin (100. mu.M) with DMSO and BDSF as reference. Therefore, the lysionotin has a good inhibiting effect on the biofilm formation of candida albicans SC 5314.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (2)
1. The application of the lysionotin in preparing the anti-candida albicans medicine is characterized in that: the anti-candida albicans drug is a drug for inhibiting the adhesion, biofilm formation and cell toxicity of candida albicans.
2. The use of nevadensin according to claim 1 in the preparation of anti-candida albicans drugs, characterized in that: the anti-candida albicans drug is a drug for preventing and/or treating candida albicans infection.
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