CN108853120B - Application of fritillaria cirrhosa in preparation of anti-candida albicans medicines - Google Patents
Application of fritillaria cirrhosa in preparation of anti-candida albicans medicines Download PDFInfo
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Abstract
The invention discloses an application of fritillaria cirrhosa in preparation of anti-candida albicans medicines. The inventor selects a compound which is high-efficiency, low-toxicity and difficult to generate drug resistance by taking the candida albicans as a test object, and finds that peiminine 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 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 peimisine in preparation of anti-candida albicans medicines.
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 provides the application of peimisine in preparing the anti-candida albicans medicine.
The purpose of the invention is realized by the following technical scheme: application of fritillaria cirrhosa in preparation of anti-candida albicans medicines.
The CAS number of the peimisine is 19773-24-1, and the structural formula is shown as follows:
specifically, the anti-candida albicans refers to inhibiting the adhesion and pathogenicity (the toxic 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) is taken as a test object, the influence of the screened peimisine on the adhesion 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 peimisine on the virulence forming factors of the Candida albicans. The result shows that peiminine has good inhibition effect on the adhesion and pathogenicity of candida albicans. Moreover, peimisine has low toxicity and does not influence the growth of human cells; meanwhile, the normal growth of the candida albicans is not influenced, which shows that the effect of peimisine 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, so that 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 fritillaria cirrhosa 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 peimisine on the pathogenicity of A549 cells of Candida albicans; wherein, the graph (A) is a graph of the cytotoxicity detection result of the fritillaria cirrhosa octyl ketone with the final concentration of 100 mu M to the A549 cells; FIG. B is a graph showing the results of measurements of various concentrations of peimisine after infecting 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 peimisine 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 peimisine on the adhesion of Candida albicans; wherein DMSO, BDSF are used as control; data shown are the average of 4 biological replicates, and error bars reflect 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 the antibacterial Activity of peimisine
1. The test method comprises the following steps:
(1) activation of candida albicans strains:
candida albicans standard strain SC5314 (also named 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 an incubator at 30 ℃.
(2) Effect of peimisine on the cellular virulence 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% CO2Cultured overnight under the conditions.
(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. Cell-waiting clothWhen the bottom of the 96-well plate was 80% full, the culture medium was discarded, and the cells were washed 3 times with 1 XPBS.
(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) the mother solution of peimisine was prepared at a concentration of 2mM by dissolving peimisine in DMSO.
b) Determination of the virulence effect of peimisine itself on cells: diluting the mother solution of peimisine to 1mM with DMSO, adding into cell maintenance solution to obtain test solution A, wherein the final concentration of peimisine is 100 μ M; meanwhile, a control group is set, namely DMSO with the same volume is used for replacing the peimisine 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 peimisine and candida albicans on cells: diluting the mother liquor of peimisine with DMSO to obtain diluted medicinal solutions with concentrations of 1mM, 500. mu.M, 250. mu.M and 125. mu.M, mixing the mother liquor of peimisine and the diluted solution of peimisine with the cell maintenance solution containing bacteria at a volume ratio of 1:9 to obtain a test solution C, wherein the final concentrations of peimisine 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; mixing BDSF mother solution (dissolved by DMSO and with the concentration of 1mM) and cell maintenance solution containing bacteria according to the volume ratio of 1:9 to obtain 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 respectively according to 100 mu L/holePlacing at 37 ℃ 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 Prism 6 treatment of the data.
(3) Determination of the Effect of peimisine 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 peimisine liquid 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 also set. Placing in a growth curve tester, measuring OD every 2h at 30 deg.C and 200rpm600Values, observed after 2d experimental results, GraphPad Prism 6 processed data.
(4) Effect of peimisine 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 selected, inoculated into GMM culture medium (6.7g/L YNB, 0.2% wt glucose), cultured overnight at 30 ℃ under shaking at 200rpm, and the OD of the bacterial solution was measured600. The cell culture broth (DMEM containing 1% vol FBS) was then used to adjust the dilution to OD6000.5. Diluting mother liquor of Bulbus Fritillariae Cirrhosae with DMSO to obtain diluted medicinal liquid with concentration of 1mM, 500 μ M, 250 μ M, 125 μ M, and mixing with above mother liquorMixing the solution and the peimisine diluent with the cell maintenance solution containing bacteria according to the volume ratio of 1:9 respectively, shaking and uniformly mixing to obtain a test solution G, wherein the final concentrations of the peimisine in the test solution G are respectively 200 mu M, 100 mu M, 50 mu M, 25 mu M and 12.5 mu M. 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 Prism 6 software.
2. Results of the experiment
(1) The peiminine has certain inhibition effect on the toxicity of the candida albicans strain SC5314
When the cytotoxicity of candida albicans is detected, the release amount of the LDH added into a DMSO group is taken as 100%, and therefore the LDH release ratio of other peimisine 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 peimisine has no toxicity to cells under the condition without Candida albicans by taking DMSO as a control, as shown in FIG. 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 peiminine has a certain protection effect on the infection of cells by inhibiting the strain SC 5314; the toxicity of Candida albicans decreased to 2.8% at 100. mu.M peiminine.
(2) Peiminine has no effect on the growth of Candida albicans strain SC5314
The results are shown in FIG. 2, where the concentration of peimisine was 100. mu.M, with DMSO as a control, had substantially no effect on the growth of Candida albicans strain SC 5314. The results indicate that peimisine does not kill bacteria and is not susceptible to drug resistance because of its action on candida albicans strain SC 5314.
(3) Adhesion of peimisine to inhibit Candida albicans strain SC5314
As shown in FIG. 3, the adhesion of Candida albicans on polystyrene was reduced by about 75.6% by DMSO and BDSF as reference, after treatment with peimine at a final concentration of 100. mu.M. The peiminine shows a certain inhibition effect on the adhesion 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 (3)
1. Application of fritillaria cirrhosa in preparation of anti-candida albicans medicines.
2. The use of peimisine according to claim 1 for the preparation of an anti-candida albicans medicament, wherein: the anti-candida albicans drug is a drug for inhibiting the adhesion and pathogenicity of candida albicans.
3. The use of peimisine according to claim 1 for the preparation of an anti-candida albicans medicament, wherein: the anti-candida albicans drug is one or two of a drug for preventing and/or treating candida albicans infection and a drug for preventing and treating infectious diseases caused by candida albicans.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1274445B1 (en) * | 1999-12-30 | 2005-09-14 | YEDA RESEARCH AND DEVELOPMENT Co. LTD. | Use of steroidal alkaloids to reverse multidrug resistance |
| CN106309455A (en) * | 2016-08-16 | 2017-01-11 | 江苏康缘药业股份有限公司 | Application of peimisine |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1274445B1 (en) * | 1999-12-30 | 2005-09-14 | YEDA RESEARCH AND DEVELOPMENT Co. LTD. | Use of steroidal alkaloids to reverse multidrug resistance |
| CN106309455A (en) * | 2016-08-16 | 2017-01-11 | 江苏康缘药业股份有限公司 | Application of peimisine |
Non-Patent Citations (1)
| Title |
|---|
| "贝母内生真菌研究进展";许勇等;《安徽农业科学》;20161231;第44卷(第28期);第141,161页 * |
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