CN110812360B - Application of resveratrol and ketoconazole in preparation of antifungal product - Google Patents
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- CN110812360B CN110812360B CN201911186984.6A CN201911186984A CN110812360B CN 110812360 B CN110812360 B CN 110812360B CN 201911186984 A CN201911186984 A CN 201911186984A CN 110812360 B CN110812360 B CN 110812360B
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- resveratrol
- candida albicans
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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
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
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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
- A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
- A61K31/05—Phenols
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/10—Antimycotics
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Abstract
The invention discloses an application of resveratrol and resveratrol combined ketoconazole in preparing a medicine for resisting fungal infection diseases. The research firstly utilizes the synergistic antifungal effect of the combination of the resveratrol and the ketoconazole, can obviously reduce the use concentration of the ketoconazole, has better bacteriostatic effect on Candida albicans, trichophyton rubrum and microsporum gypseum than that of the two substances which are used independently, has the bacteriostatic rate of over 90 percent, obviously improves the bacteriostatic rate, and has wide development and application prospects through the application of the combination of the resveratrol and the ketoconazole in the preparation of the antifungal infection disease medicines.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to the technical field of application of resveratrol and ketoconazole in preparation of antifungal products.
Background
Fungi are a class of eukaryotic organisms that exist in nature, are widely diverse and distributed, and can infect different parts of the human body and animals. Fungal infections can be divided into two categories, superficial and deep fungal infections: superficial fungal infection is usually caused by dermatophyte infection, which infects epidermis, hands and feet, hair, nails and other parts, generally mainly shows tinea, dermatitis and the like, is difficult to cure and is easy to recur; deep fungal infections include subcutaneous tissue infections and systemic fungal infections that can infect the human subcutaneous tissue, internal organs, blood, bones and central nervous system, and can severely cause death. In recent years, the incidence and mortality of fungal infections have been rising dramatically, and these diseases have become one of the major health-threatening diseases, especially fatal injuries to patients with functional immune defects such as organ transplantation, cancer, AIDS, etc. Candida albicans is the most common pathogenic fungus in clinic, and about more than half of fungal infections are caused by Candida albicans.
Currently, clinically used antifungal drugs can be divided into: ketoconazoles, allylamines, echinocandins, azoles tetrazoles. Azole medicine is the first choice medicine for clinical treatment and prevention of Candida albicans, and plays a great role in controlling pathogenic fungus infection. However, the large application of azole drugs enables candida which are sensitive to azoles to continuously generate drug resistance, and brings great challenge to clinical antifungal infection. In the face of the problem of drug effect weakening caused by multi-drug resistant fungi, high doses of drugs are required to achieve the treatment effect, so that severe nephrotoxicity and hepatotoxicity are caused, and the physical health of people is seriously influenced. The combined application of antifungal medicines is concerned at present, the dosage and the administration frequency of the medicines can be reduced, the adverse reaction of the medicines is reduced, the antibacterial range is expanded, and the antifungal effect of the medicines is improved.
The combined application of the resveratrol and the ketoconazole provided by the invention can enhance the effect and reduce the toxicity, the combined application of the resveratrol and the ketoconazole shows important application values in medicines, health care products and beauty products, and no report is found on the research of preparing antifungal products by using the combined application of the resveratrol and the ketoconazole medicines at present.
Disclosure of Invention
Aiming at the technical current situation that the combined application of resveratrol and ketoconazole to prepare antifungal products is not recorded in the prior art, the screening of the antifungal drug combined application can reduce the dosage and the administration times, reduce the adverse drug reaction, enlarge the antibacterial range and improve the antifungal effect of the drug is concerned at present. The invention aims to: the problem of great toxic and side effects of the existing fungi drug resistance and antifungal drugs is solved, and the antifungal drug composition is provided, wherein resveratrol and ketoconazole are used as main active ingredients of the antifungal drug composition. The resveratrol which has the synergistic antifungal effect with ketoconazole is separated from the myxobacteria metabolite by utilizing an interactive screening model for the first time, the use concentration of the ketoconazole can be obviously reduced, the defect that the resveratrol almost has no Candida albicans resistance is overcome, and the bacteriostasis rate can be obviously improved.
The invention provides an application of resveratrol and ketoconazole in preparation of antifungal products.
Preferably, the fungus is trichophyton rubrum or candida albicans or microsporum gypseum.
Preferably, the resveratrol is preferably produced by fermentation production from a myxobacteria (Myxococcus virescens).
Preferably, the fermentation medium adopted by the myxobacteria (Myxococcus virescens) is HT medium: 3.0% of soluble starch, 0.1% of dipotassium phosphate, 0.01% of calcium chloride, 0.015% of magnesium sulfate heptahydrate, 0.001% of ferrous chloride, 0.25% of sodium nitrate and pH 7.2.
The invention provides an application of resveratrol and ketoconazole in preparation of antifungal products, wherein when the products for resisting candida albicans are prepared, the usage amount of resveratrol is 50-400 mu g/mL, and the usage amount of ketoconazole is 0.09-22.5 mu g/mL.
Preferably, when preparing the anti-Candida albicans product, the optimal usage amount of the resveratrol is 200 mug/mL, and the usage amount of the ketoconazole is 11.25 mug/mL.
The invention provides an application of resveratrol and ketoconazole in preparation of antifungal products, wherein when the product for resisting trichophyton rubrum is prepared, the usage amount of resveratrol is 200 mu g/mL, and the usage amount of ketoconazole is 0.39-0.0551 mu g/mL.
Preferably, when preparing the product for resisting trichophyton rubrum, the optimal usage amount of the resveratrol is 200 mu g/mL, and the usage amount of the ketoconazole is 0.19 mu g/mL.
The invention provides an application of resveratrol and ketoconazole in preparation of antifungal products, wherein when the products for resisting microsporidia gypsea are prepared, the usage amount of resveratrol is 200 mug/mL, and the usage amount of ketoconazole is 0.78-0.011 mug/mL.
Preferably, when preparing products for resisting microsporidian gypseum, the optimal usage amount of the resveratrol is 200 mug/mL, and the usage amount of the ketoconazole is 0.39-0.78 mug/mL.
The myxobacteria (Myxococcus virescens) adopted by the invention can be purchased by public through a well-known strain preservation unit or other public channels to implement the invention.
By implementing the specific technical scheme provided by the invention, the following beneficial effects can be achieved:
(1) The strain myxobacteria (Myxococcus virescens) adopted by the invention has the characteristics of higher fermentation production of resveratrol, and has the characteristics of simple culture condition and rapid propagation.
(2) The invention discloses an application of resveratrol and resveratrol combined ketoconazole in preparing a medicine for resisting fungal infection diseases. When the resveratrol and the ketoconazole are jointly used, the bacteriostatic effects on candida albicans, trichophyton rubrum and microsporum gypseum are better than those of two substances which are singly used under the same using concentration, wherein when the two substances are singly acted on the candida albicans, the half bacteriostatic concentrations of the resveratrol and the ketoconazole on the candida albicans are 3200 mug/mL and 45 mug/mL respectively, when the resveratrol and the ketoconazole are jointly acted on the candida albicans, the resveratrol concentration is 200 mug/mL, the ketoconazole concentration is 0.18 mug/mL, and the bacteriostatic rate on the candida albicans can reach more than 50%; the resveratrol concentration is 200 mug/mL, the ketoconazole concentration is 11.25 mug/mL, and the bacteriostasis rate to candida albicans can reach more than 95%. When the resveratrol and the ketoconazole act on the trichophyton rubrum independently, the half inhibitory concentrations of the resveratrol and the ketoconazole to the trichophyton rubrum are 400 mu g/mL and 0.045 mu g/mL respectively, and when the resveratrol and the ketoconazole act on the trichophyton rubrum jointly, the resveratrol concentration is 200 mu g/mL, the ketoconazole concentration is 0.011 mu g/mL, and the inhibitory rate to the trichophyton rubrum can reach more than 50%; the resveratrol concentration is 200 mug/mL, the ketoconazole concentration is 0.09 mug/mL, and the bacteriostasis rate to trichophyton rubrum can reach more than 95%. When the resveratrol and the ketoconazole act on the microsporidia gyelike independently, the half bacteriostatic concentrations of the resveratrol and the ketoconazole to the microsporidia gyelike are respectively 150 mu g/mL and 0.39-0.78 mu g/mL, and when the resveratrol and the ketoconazole act on the microsporidia gyelike jointly, the resveratrol concentration is 200 mu g/mL, the ketoconazole concentration is 0.39 mu g/mL, and the bacteriostatic rate to the microsporidia gyelike can reach more than 91 percent; therefore, the natural resveratrol is used for replacing part of ketoconazole to inhibit fungi, so that the using amount of the ketoconazole can be reduced, and the research and the development of novel, efficient and natural antifungal infection medicines are facilitated.
Drawings
FIG. 1 shows the HPLC fractions of metabolites of a strain of myxobacteria.
FIG. 2 shows the 1H-NMR and molecular structure of Compound 1.
Fig. 3 is a graph showing the effect of the combination of resveratrol and ketoconazole on the growth curve of candida albicans.
Fig. 4 is a graph showing the bacteriostatic effect of the combined application of resveratrol and ketoconazole on trichophyton rubrum.
FIG. 5 shows the bacteriostatic effect of the combined application of resveratrol and ketoconazole on microsporidian gypsea
FIG. 6 is a graph showing the bacteriostatic rate of six drugs at different concentrations when applied alone or in combination with resveratrol.
Wherein, the A picture is amphotericin B, the B picture is miconazole, the C picture is itraconazole, the D picture is flucytosine, the E picture is fluconazole, and the F picture is ketoconazole.
Detailed Description
The present invention will be described below by way of examples, but the present invention is not limited to the following examples. All raw and auxiliary materials selected for use in the present invention, as well as methods for culturing the selected bacterial species, are well known and used in the art, and all percentages referred to herein are by weight unless otherwise indicated.
The myxobacteria (Myxococcus virescens) adopted by the invention belongs to common myxobacteria, and the myxobacteria can be purchased by public through a well-known strain preservation unit or other public channels to implement the invention.
In the examples of the present invention, the following basic medium was used for myxobacteria (Myxococcus virescens): HT medium, 3.0% of soluble starch, 0.1% of dipotassium phosphate, 0.01% of calcium chloride, 0.015% of magnesium sulfate heptahydrate, 0.001% of ferrous chloride, 0.25% of sodium nitrate and pH 7.2.
The following reagents and materials are adopted in the embodiment of the invention: HP20 macroporous resin, water, methanol, dimethyl sulfoxide (DMSO), sasa's broth culture medium, ketoconazole, potato solid culture medium, amphotericin B, miconazole, itraconazole, flucytosine, fluconazole
The first embodiment is as follows: application of resveratrol and ketoconazole in preparation of antifungal product
The invention provides an application of resveratrol and ketoconazole in preparation of antifungal products.
Preferably, the fungus is trichophyton rubrum or candida albicans or microsporum gypseum.
The resveratrol is preferably prepared by fermentation production metabolism by myxobacteria (Myxococcus virescens).
The invention provides an application of resveratrol and ketoconazole in preparation of antifungal products, wherein when the resveratrol and ketoconazole are used as candida albicans resistant products, the usage amount of resveratrol is 50-400 mu g/mL, and the usage amount of ketoconazole is 0.09-22.5 mu g/mL.
Preferably, when the resveratrol derivative is used as an anti-Candida albicans product, the optimal use amount of the resveratrol is 200 mug/mL, and the use amount of the ketoconazole is 11.25 mug/mL.
The invention provides an application of resveratrol and ketoconazole in preparation of antifungal products, wherein when the resveratrol and ketoconazole are used as anti-trichophyton rubrum products, the usage amount of resveratrol is 200 mug/mL, and the usage amount of ketoconazole is 0.39-0.0055 mug/mL.
Preferably, when the product is used as an anti-trichophyton rubrum product, the optimal usage amount of the resveratrol is 200 mu g/mL, and the usage amount of the ketoconazole is 0.045 mu g/mL.
The invention provides an application of resveratrol and ketoconazole in preparing antifungal products, wherein when the resveratrol and ketoconazole are used as products for resisting microsporidia gypsea, the usage amount of resveratrol is 200 mug/mL, and the usage amount of ketoconazole is 0.78-0.011 mug/mL.
Preferably, when the resveratrol compound is used as a product for resisting microsporidian gypseum, the optimal use amount of the resveratrol is 200 mu g/mL, and the use amount of the ketoconazole is 0.39-0.78 mu g/mL.
Example two: preparation of resveratrol
The experimental strain is purchased Myxococcus viscosus (Myxococcus virescens), cultured by a fermentation medium HT medium, filtered by fermentation liquor, adsorbed by HP20 macroporous resin, and sequentially eluted by water and methanol with the volume of 10 times of the resin respectively. The methanol eluent is distilled under reduced pressure at 50 ℃ to remove the solvent, thus obtaining a methanol crude extract. Subjecting the crude methanol extract to silica gel column chromatography, CHCl 3 MeOH gradient elution, one collection unit per 100mL of eluate, TLC check of elution progress. Ketoconazole and each component are used for carrying out interactive activity tracking screening on candida albicans, several sections with better activity are selected for separation and purification, and finally, a preparation liquid phase is used for collecting an active monomer compound. The structural identification of the compound was carried out by 1H-NMR. The results of the identification are shown in FIG. 1. TLC shows that two yellowish single spots with different polarities are obtained, namely a compound 1 and a compound 2, wherein the compound 1 has better interactive activity, and the structure of the spot is further identified. The results are shown in FIG. 2 and Table 1.
Table 1: NMR data of Compound 1
As shown in figure 2 and table 1, through analysis of compound spectrogram data and combination of necessary physicochemical means and reference of literature, compound 1 is identified to be trans-resveratrol, and the molecular formula is C 14 H 12 O 3 Molecular weight 228.24, chemical name 3,4', 5-trihydroxy stilbene.
Example three: half inhibitory concentration (MIC) of resveratrol on Candida albicans 50 ) Detection of
Based on example two, the compound 1 trans-resveratrol prepared above was dissolved in 100% dimethyl sulfoxide (DMSO) to prepare a resveratrol sample solution with a concentration of 20mg/mL, and the resveratrol sample solution was added to the saybolt broth culture medium by two-fold dilution method to obtain resveratrol concentrations of 8000, 4000, 2000, 1000, 500, 250 and 125 μ g/mL. Taking a 96-well bacterial culture plate, and adding 100 mu L of 10-concentration bacterial culture plate into each well 5 CFU/mL Candida albicans solution and 100 mu L resveratrol sample solution with each concentration, and setting a blank control (culture medium without resveratrol sample solution + bacteria solution) and a solvent control (DMSO solution + bacteria solution), and setting a control culture plate (without bacteria solution). Culturing for 24h in a 30-degree incubator, taking out a 96-hole culture plate, placing the culture plate in an enzyme labeling instrument, linearly oscillating for 30s, detecting a light absorption value at 630nm, and passing through a formula according to experimental data: bacteriostatic rate (%) = (1-determination hole OD630nm value/blank control hole OD630nm value) × 100% the bacteriostatic rate of resveratrol on Candida albicans at each concentration was calculated, and the results are shown in Table 2.
Table 2: bacteriostasis rate of resveratrol with different concentrations on candida albicans
| Different concentration groups | Concentration of μ g/mL | Bacteriostatic ratio (%) |
| |
0 | 0 |
| 1 | 62.5 | -4.15％ |
| 2 | 125 | -5.05％ |
| 3 | 250 | -1.13％ |
| 4 | 500 | 3.42％ |
| 5 | 1000 | 5.74％ |
| 6 | 2000 | 15.57％ |
| 7 | 4000 | 83.69％ |
As can be seen from Table 2, low concentrations of resveratrol had little inhibitory effect on Candida albicans. As the concentration rises to over 1000 mug/mL, the inhibitory activity of the compound on the candida albicans begins to appear, and the inhibitory rate can be obtained, wherein the half inhibitory concentration MIC of the compound 50 The value was 3200. Mu.g/mL. Therefore, resveratrol at concentrations below 1000. Mu.g/mL had little bacteriostatic activity against Candida albicans.
Example four: median Inhibitory Concentration (MIC) of ketoconazole on Candida albicans 50 ) Detection of
Ketoconazole is dissolved in 100% dimethyl sulfoxide (DMSO) to prepare a ketoconazole sample solution with the concentration of 10mg/mL, and the ketoconazole sample solution is added into a Sasa's broth culture medium by a twofold dilution method until the concentrations of the ketoconazole are respectively 200, 100, 50, 25, 12.5, 6.25, 3.13, 1.56, 0.78 and 0.39 mu g/mL. Adding 100 μ L of 10-concentration bacteria culture plate into each well 5 CFU/mL Candida albicans solution and 100 μ L ketoconazole sample solution with each concentration, so that the final concentration of ketoconazole is 100, 50, 25, 12.5, 6.25, 3.13, 1.56, 0.78, 0.39 and 0.19 μ g/mL respectively. Setting blank control (culture medium without ketoconazole sample solution + bacteria solution) and solvent control (DMSO solution + bacteria solution), and setting control culture plate(s) ((ii) (culture medium without ketoconazole sample solution) + (bacteria solution))No added bacteria solution). Culturing for 24h in a 30-degree incubator, taking out a 96-hole culture plate, placing the culture plate in an enzyme labeling instrument, linearly oscillating for 30s, detecting a light absorption value at 630nm, and passing through a formula according to experimental data: bacteriostatic rate (%) = (1-determination hole OD630nm value/blank control hole OD630nm value) × 100% the bacteriostatic rate of ketoconazole to candida albicans at each concentration was calculated, and the results are shown in table 3.
Table 3: bacteriostasis rate of ketoconazole sample liquid with different concentrations on candida albicans
As shown in Table 3, the inhibition rate of ketoconazole on Candida albicans has concentration dependence, the inhibition activity of ketoconazole on Candida albicans is gradually enhanced with the increase of the concentration, and the inhibition rate can be obtained as the MIC of ketoconazole with half inhibitory concentration 50 The value was between 25 and 50. Mu.g/mL, calculated as 45. Mu.g/mL.
Example five: research on bacteriostatic action of resveratrol and ketoconazole on candida albicans
MIC of resveratrol against Candida albicans, determined as described above, based on examples three and four 50 Adding 20mg/ml resveratrol sample solution into Sabouraud's broth culture medium by double dilution method until the concentration of resveratrol in each sample solution is 1/2MIC 50 、1/4MIC 50 、1/8MIC 50 And 1/16MIC 50 Namely 1600. Mu.g/mL, 800. Mu.g/mL, 400. Mu.g/mL, 200. Mu.g/mL. According to the MIC of the ketoconazole to the Candida albicans determined above, 10mg/ml of ketoconazole sample solutions were added to the Sasa's broth by the double dilution method until the concentration of the ketoconazole in each sample solution was 2MIC 50 、MIC 50 、1/2MIC 50 、1/4MIC 50 、1/8MIC 50 、1/16MIC 50 、1/32MIC 50 、1/64MIC 50 And 1/128MIC 50 . Taking 96-well bacterial culture plate, adding into each well50 μ L of resveratrol and ketoconazole solutions diluted in culture medium at each concentration, and then 100 μ L of 10 concentration solution was added to each well 5 CFU/ml Candida albicans solution, and blank control (culture medium without resveratrol and ketoconazole sample solution + bacteria solution), solvent control (DMSO + bacteria solution), and control culture plate (without bacteria solution) are set. Culturing for 24h in a 30-degree incubator, taking out a 96-hole culture plate, placing the culture plate in a microplate reader, linearly oscillating for 30s, detecting a light absorption value at 630nm, and passing through a formula according to experimental data: bacteriostatic rate (%) = (1-determination hole OD630nm value/blank control hole OD630nm value) × 100% calculating the bacteriostatic rate of resveratrol and ketoconazole on Candida albicans at each concentration. The experiment is set to be 3 times, the effect of the combined administration of the ketoconazole and the resveratrol is evaluated by grading bacteriostatic concentration indexes and combining a chessboard method, and the FICI index of the combined ketoconazole and resveratrol is calculated by the following formula:
FICI＝MIC a association /MIC a alone +MIC b in combination with /MIC b alone
The judgment standard is as follows: if the FICI is less than or equal to 0.5, the synergistic effect is obtained; if the 0.5-woven fabric FICI is less than or equal to 1, the additive effect is achieved; when FICI is greater than 4, antagonism is achieved; when 1 instead of FICI is less than or equal to 4, no relevant effect is achieved.
The results of the calculation of the bacteriostatic rate are shown in table 4.
TABLE 4 chessboard method for showing the bacteriostasis rate of the combined medicine of ketoconazole and resveratrol against Candida albicans
As can be seen from Table 4, MIC of Candida albicans when Ketoconazole and resveratrol were used in combination 50 The value decreases significantly. The concentration of ketoconazole is 1/256MIC 50 (0.18 mu g/mL) and the resveratrol concentration is 1/16MIC 50 The bacteriostasis rate to candida albicans reaches more than 50 percent when the antibacterial agent is 200 mug/mL. FICI =1/256+1/16=0.066. Namely, the ketoconazole and the resveratrol have synergistic antibacterial effect on the candida albicans when being used together. Of particular note is the concentration of ketoconazole at 100 μ g/mL versus white ink when ketoconazole is used aloneThe bacteriostasis rate of the pearl bacteria is only about 56 percent, but 1/4MIC is used in combined application 50 (11.25. Mu.g/mL) ketoconazole and 1/16MIC 50 The (200 mu g/mL) resveratrol has the bacteriostasis rate of over 95 percent on the Candida albicans. Therefore, the ketoconazole and the resveratrol have a synergistic antibacterial effect on the candida albicans when being used in a combined way, and the effect of resisting the candida albicans is very obvious when being used in a combined way.
Example six: effect of resveratrol and Ketoconazole combination on Candida albicans growth Curve
A96-well bacterial culture plate is taken, and 50 mu L of 800 mu g/mL resveratrol sample solution diluted by the culture medium and 50 mu L of the culture medium are respectively added into the first row to ensure that the final concentration is 200 mu g/mL. The second column was filled with 50. Mu.L of the medium-diluted sample solution of ketoconazole at 45. Mu.g/mL and 50. Mu.L of the medium, respectively, to a final concentration of 11.25. Mu.g/mL, and the third column was filled with 50. Mu.L of the medium-diluted sample solution of resveratrol at 800. Mu.g/mL and 50. Mu.L of the medium-diluted sample solution of ketoconazole at 45. Mu.g/mL, respectively, to a final concentration of 200. Mu.g/mL and 11.25. Mu.g/mL, respectively. The fourth column is blank. Then 100. Mu.L of 10 concentration was added to each well 5 CFU/ml Candida albicans solution. Culturing 96-well culture plate in microplate reader at 30 deg.C, continuously monitoring for 30h, linearly oscillating for 30s before each detection, and detecting light absorption value at 630 nm. The influence of the combined application of resveratrol and ketoconazole on the growth curve of Candida albicans is studied. The measurement results are shown in FIG. 3. The bacteriostasis rate of the ketoconazole and the resveratrol which are jointly applied (the concentration is respectively 11.25 mug/mL and 200 mug/mL) to the candida albicans can reach more than 95 percent. When the ketoconazole is used alone, the inhibition rate of the ketoconazole concentration of 100 mu g/mL on the candida albicans is only about 56%. 1/4MIC 50 The combined application of the ketoconazole and the resveratrol has obvious bacteriostasis effect, the bacteriostasis rate on the candida albicans can reach more than 95 percent, and the action on the candida albicans is changed from bacteriostasis to sterilization.
Example seven: research on bacteriostasis of clinical candida albicans by combined application of resveratrol and ketoconazole
Based on the second to sixth embodiments, 30 clinical Candida albicans strains were obtained from Min hospitals in Shaanxi province. After activation, the combined resveratrol and ketoconazole combined application is detected to inhibit 30 clinical candida albicans according to the method of content 4, and the FICI value is calculated. The FICI values of the respective clinical strains are tabulated in table 5.
Table 5: bacteriostatic action of combined application of resveratrol and ketoconazole on clinical candida albicans
From the above results, it was found that 24 strains showed synergistic effects, 4 strains showed additive effects, and 2 strains showed no synergistic effects among 30 test strains of different origins. Therefore, the combined application of the resveratrol and the ketoconazole has obvious synergistic bacteriostatic action on clinically separated candida albicans. In Candida albicans obtained by clinical separation, the strain with synergistic effect accounts for more than 80%.
Example eight: bacteriostatic effect of combined application of resveratrol and ketoconazole on trichophyton rubrum
Based on the second to fourth examples, trichophyton rubrum was activated on potato/potato solid medium and cultured in a 28-degree incubator for 7d. And then continuing to activate once, after culturing for 7d, adding 4ml of sterile physiological saline to the solid plate, and gently scraping the spores by using a sterile spatula to dissolve in the physiological saline to obtain spore suspension. After standing for 3min, transferring the upper spore suspension into a sterile centrifuge tube, and carrying out vortex oscillation for 20s. Filtering with sterile double-layer gauze, and removing mycelium to obtain final spore suspension. Spore suspensions were counted by a hemocytometer. Diluting spore solution to 10% with potato and potato culture medium 5 CFU/ml. Ketoconazole is dissolved in 100% dimethyl sulfoxide (DMSO) to prepare a ketoconazole sample solution with the concentration of 10mg/mL, and the ketoconazole sample solution is added into a potato culture medium by a double dilution method until the concentrations of the ketoconazole are respectively 0.78, 0.39, 0.19, 0.09, 0.045, 0.022 and 0.011 mu g/mL. Taking 96-well bacteriaPlates were incubated at 10 concentration in 100. Mu.L/well 5 CFU/mL Trichophyton rubrum spore solution and 100 μ L ketoconazole sample solution with each concentration, so that the final concentrations of ketoconazole are 0.39, 0.19, 0.09, 0.045, 0.022, 0.011 and 0.0055 μ g/mL respectively. And a blank control (culture medium without ketoconazole sample solution + bacteria solution) and a solvent control (DMSO solution + bacteria solution) are set, and a control culture plate (without bacteria solution) is set at the same time. Placing a 96-hole culture plate in an enzyme labeling instrument for 30-degree culture, continuously monitoring for 7d, linearly oscillating for 30s before each detection, detecting a light absorption value at 630nm, and passing through a formula according to experimental data: the inhibition rate (%) is not = (1-determination hole OD630nm value/blank control hole OD630nm value) × 100%, the inhibition rate of each concentration of ketoconazole to trichophyton rubrum is calculated, and the MIC is calculated 50 . In addition, 50 mu L of resveratrol sample solution diluted by a culture medium and with the final concentration of 200 mu g/mL and ketoconazole sample solution with different concentrations are added into each hole of a 96-hole plate respectively, blank control and solvent control are set, the culture is carried out by the same method, the absorbance at 630nm is detected, and the bacteriostatic effect of the combined application of the resveratrol and the ketoconazole on trichophyton rubrum is detected. The bacteriostatic effect of resveratrol and ketoconazole on trichophyton rubrum in combination is shown in figure 4 and table 6.
Table 6: inhibition rate of ketoconazole with different concentrations on trichophyton rubrum independently and in combination with resveratrol
| Different concentration groups | At a concentration of. Mu.g/mL | Single rate of inhibition of bacteria | Combined rate of inhibition of |
| Blank space | |||
| 0 | 0 | 36.94％ | |
| 1 | 0.0055 | 7.94％ | 42.46％ |
| 2 | 0.011 | 21.29％ | 58.92％ |
| 3 | 0.022 | 29.11％ | 67.78％ |
| 4 | 0.045 | 51.90％ | 72.38％ |
| 5 | 0.09 | 69.85％ | 96.55％ |
| 6 | 0.19 | 84.12％ | 98.62％ |
| 7 | 0.39 | 97.12％ | 98.39％ |
As is clear from Table 6 and FIG. 4, the inhibitory rate of ketoconazole on Trichophyton rubrum is concentration-dependent, and the inhibitory activity on Trichophyton rubrum is gradually increased with increasing concentration, and the inhibitory rate is shown as MIC 50 The value was about 0.045. Mu.g/mL. Meanwhile, the bacteriostasis rate is 36.94 percent when only 200 mug/mL of resveratrol is added. The resveratrol has a certain inhibiting effect on trichophyton rubrum when being singly applied, the inhibiting rate is improved when the resveratrol and the ketoconazole are jointly applied, and the inhibiting effect of the joint application is an additive effect. Therefore, the resveratrol and the ketoconazole have additive bacteriostatic action on trichophyton rubrum when being used together.
Example nine: bacteriostatic effect of combined application of resveratrol and ketoconazole on microsporidia gypsea
Based on example eight, only the ketoconazole sample solutions were changed to final concentrations of 0.78, 0.39, 0.19, 0.09, 0.045, 0.022, 0.011 μ g/mL. To detect the bacteriostatic effect of the combined application of resveratrol and ketoconazole on microsporidian gypseum. The statistical results of the bacteriostatic rate are shown in figure 5 and table 7.
Table 7: inhibition rate of ketoconazole with different concentrations on microsporidia gypsea alone and in combination with resveratrol
| Different concentration groups | Concentration of μ g/mL | Single rate of inhibition of bacteria | Combined rate of inhibition of |
| Blank space | |||
| 0 | 0 | 63.0％ | |
| 1 | 0.011 | 1.0％ | 64.7％ |
| 2 | 0.022 | 2.1％ | 66.4％ |
| 3 | 0.045 | 8.9％ | 72.5％ |
| 4 | 0.09 | 17.4％ | 75.6％ |
| 5 | 0.19 | 28.1％ | 78.9％ |
| 6 | 0.39 | 35.4％ | 91.4％ |
| 7 | 0.78 | 62.4％ | 97.1％ |
As can be seen from Table 7 and FIG. 5, the inhibition rate of ketoconazole on microsporidian gypseudum is concentration-dependent, and the inhibition activity of ketoconazole on microsporidian gypseudum is gradually enhanced with the increase of the concentration, and the inhibition rate is determined as the MIC of ketoconazole 50 The value is between 0.39 and 0.78. Mu.g/mL. Meanwhile, when the resveratrol with the concentration of 200 mu g/mL is singly applied, the bacteriostasis rate is 63 percent. Therefore, the resveratrol has obvious inhibition effect on the microsporidia gypseum, the inhibition rate is improved when the resveratrol and the ketoconazole are jointly applied, and the combined inhibition effect is additive effect. The resveratrol and ketoconazole have additive bacteriostatic action on gypsum-like microsporidian when being used together.
Example ten: research on bacteriostatic effect of resveratrol and other antifungal drugs on candida albicans
Based on the second, third and fifth examples, the bacteriostatic effect of the combination of five other antifungal drugs and resveratrol on candida albicans was determined. The five medicines comprise: amphotericin B, miconazole, itraconazole, flucytosine, and fluconazole. Calculating MIC of six drugs 50 The value is obtained. Study of 1/2MIC 50 、1/4MIC 50 、1/8MIC 50 The bacteriostatic rate of the concentration drug when being used alone or combined with resveratrol of 200 mug/mL. The measurement results are shown in FIG. 6. The bacteriostatic rate of resveratrol and six drugs with different concentrations is shown in figure 6. The left side shows the bacteriostatic rate of 3 drugs with different concentrations when used alone, and the right side shows the bacteriostatic rate of 3 drugs with the same concentration when used together with resveratrol. Therefore, the combined application of the resveratrol and the ketoconazole has the advantages that the bacteriostasis rate is obviously improved, the resveratrol and the ketoconazole have obvious synergistic antibacterial action, certain synergistic antibacterial action is realized when the resveratrol and the ketoconazole are combined with the miconazole, and no obvious interaction exists between the resveratrol and the ketoconazole and the amphotericin B, flucytosine, fluconazole and itraconazole.
The above examples are merely illustrative for clearly illustrating the present invention and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. This need not be, nor should it be exhaustive of all embodiments. And obvious variations or modifications can be made while remaining within the scope of the present invention.
Claims (2)
1. An application of resveratrol and ketoconazole in preparing antifungal products is characterized in that fungi are Candida albicans; the resveratrol is obtained by fermentation and metabolism production of myxobacteria (Myxococcus virescens); when the product for resisting candida albicans is prepared, the using amount of resveratrol is 50-400 mu g/mL, and the using amount of ketoconazole is 0.09-22.5 mu g/mL.
2. The use of resveratrol in combination with ketoconazole in the preparation of antifungal products as in claim 1, wherein the resveratrol is used in an optimum amount of 200 μ g/mL and the ketoconazole is used in an optimum amount of 11.25 μ g/mL when preparing products against candida albicans.
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