WO2023019924A1

WO2023019924A1 - Use of caffeol or derivative thereof in preparation of anti-candida-albicans drug or anti-candida-albicans daily articles

Info

Publication number: WO2023019924A1
Application number: PCT/CN2022/080168
Authority: WO
Inventors: 邓音乐; 宋施豪; 王歌润; 谭慧慧
Original assignee: 中山大学
Priority date: 2021-08-20
Filing date: 2022-03-10
Publication date: 2023-02-23
Also published as: CN113648303A; CN113648303B

Abstract

Provided in the present invention is the use of caffeol or a derivative thereof in the preparation of an anti-Candida-albicans drug or anti-Candida-albicans daily articles. According to the present invention, a compound with high efficiency, low toxicity and not prone to developing drug resistance is selected by taking Candida albicans as a test object. It is found that caffeol or a derivative thereof has a good inhibitory effect on the adhesion, hypha formation, biofilm formation and pathogenicity of Candida albicans. Moreover, kahweol has a good inhibitory effect on the infection of a Candida albicans strain SC5314 on a mouse oral cavity, and the compound itself is not toxic to human cells. Low-concentration kahweol and cafestol have no inhibitory effect on the cell growth of Candida albicans, this indicates that the effect of caffeol or the derivative thereof involves inhibiting the expression of pathogenic factors of Candida albicans instead of relying on killing Candida albicans cells, and thus, drug resistance does not tend to develop.

Description

Application of cafestol or its derivatives in the preparation of anti-candida albicans medicine or anti-candida albicans daily necessities

technical field

The invention belongs to the technical field of biomedicine, and particularly relates to the application of cafestol or derivatives thereof in preparing anti-candida medicaments or anti-candida daily necessities.

Background technique

Candida albicans (Candida albicans) is a fungal disease widely spread in humans. It is an important opportunistic pathogenic fungus, usually causing acute, subacute or chronic infections, and it is also one of the most important pathogens of hospital-acquired infections. . On healthy human mucosal surfaces, such as the oral cavity and intestinal tract, Candida albicans usually does not cause disease, but in patients with compromised or suppressed immune systems, such as chemotherapy patients, organ transplant patients or AIDS patients, it can cause serious systemic damage. Sexual infection, the fatality rate is as high as 40%.

At present, there are limited types of antifungal drugs clinically, among which azole drugs (fluconazole) are widely used, and fluconazole plays a bacteriostatic effect by inhibiting fungal replication, but with the abuse of antibiotics, the phenomenon of drug resistance is becoming more and more serious. It's getting serious.

Contents of the invention

The purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art, and provide the application of cafestol or its derivatives in the preparation of anti-Candida albicans medicine or anti-Candida albicans daily necessities.

The object of the present invention is achieved through the following technical scheme: the application of cafestol or its derivatives in the preparation of anti-Candida albicans medicine or anti-Candida albicans daily necessities.

The derivative is preferably kahweol, the CAS number is 6894-43-5, and its structural formula is as follows:

Described cafestol, also known as coffee oil alcohol, CAS number is 469-83-0, and its structural formula is as follows:

Specifically, the anti-Candida albicans refers to inhibiting the adhesion, mycelia formation, biofilm formation, and pathogenicity of Candida albicans (infection and pathogenicity of Candida albicans).

The anti-Candida medicaments include medicaments for preventing and/or treating Candida albicans infection, and medicaments for preventing and/or treating infectious diseases caused by Candida albicans.

The daily necessities are preferably mouthwash or vaginal lotion.

The present invention has the following beneficial effects:

Yeast-mycelium diphasia is a unique characteristic of Candida albicans. The free yeast state is not pathogenic to the host during infection, and it mainly performs the role of adhering to the recipient tissue, and then undergoes a transformation from yeast state to mycelial state to promote invasion, and then invades in the form of mycelium Infect the host tissue and further play the role of virulence. Yeast-hyphae morphological transformation is an important process for Candida albicans to exert virulence. Therefore, in the previous research work of the present invention, starting from the unique yeast-hyphae duality of Candida albicans, targeted screening of compounds with high efficiency, low toxicity, and less resistance to drugs. Then with Candida albicans (Candida albicans) as test object, investigated respectively the influence of kahwegol and cafeteria alcohol screened by the present invention on the adhesion, hyphal formation rate, biofilm formation and cytotoxicity of Candida albicans The purpose is to further affect the infection of Candida albicans by detecting the interference of cafestol and cafestyl alcohol on the virulence factors of Candida albicans. The results showed that high concentrations of kahweol had a slight inhibitory effect on the growth of Candida albicans, low concentrations of kahweol and cafestyl alcohol had no inhibitory effect on the growth of Candida albicans, and they all had the ability to adhere to Candida albicans , mycelia formation, biofilm formation and pathogenicity have very good inhibitory effects, indicating that the inhibitory effect of cafestol and cafestyl alcohol on the virulence of Candida albicans is not mainly by killing Candida albicans cells, but by Inhibit the adhesion, mycelia formation, biofilm formation and pathogenicity of Candida albicans, so it is not easy to produce drug resistance. Moreover, cafestol and cafestyl alcohol do not affect human cells and are non-toxic to human cells. Therefore, kahweol and cafestyl alcohol have good application prospects in the development of new antifungal drugs, especially the development of anti-Candida albicans infection drugs

Therefore, the application of cafestyl alcohol and cafestyl alcohol in the preparation of medicines against Candida albicans infection, and the application of medicines in the preparation of prevention and/or treatment of infectious diseases caused by Candida albicans should be protected under the protection of the present invention. within range.

Description of drawings

Figure 1 is a photomicrograph showing that kahweol and kahweol at a final concentration of 30 μg/mL respectively inhibit the formation of Candida albicans hyphae; wherein, a DMSO control group was set.

Figure 2 is a graph showing the measurement results of cafestol and cafestyl alcohol respectively inhibiting the hyphae formation rate of Candida albicans; this data shows the average results of three biological experiments, and the error bars reflect the standard deviation.

Figure 3 is a graph showing the results of cafestol and cafestyl alcohol respectively inhibiting the adhesion of Candida albicans; wherein, DMSO was used as a control group; the data shows the average results of 4 biological repetitions, and the error bars reflect the standard deviation.

Figure 4 is a graph showing the results of cafestol and cafestyl alcohol respectively inhibiting the formation of Candida albicans biofilm; wherein, DMSO was used as a control group; the data shows the average results of 4 biological repetitions, and the error bars reflect the standard deviation.

Figure 5 is a graph showing the effects of cafestol and cafestyl alcohol on the growth rate of Candida albicans respectively; wherein, DMSO is used as a control; the data shows the average results of 3 biological repetitions, and the error bars reflect the standard deviation.

Figure 6 is a graph showing the effects of kahweol and kahweol on the toxicity of Candida albicans on A549 cells; among them, Figure A is the cytotoxicity detection of kahweol and kahweol at a final concentration of 30 μg/mL on A549 cells Result chart; Figure B is the detection result chart after different concentrations of kahweol and kahweol inhibit Candida albicans from infecting cells.

Fig. 7 is a graph showing the results of detection of cafestol on oral cavity infection of Candida albicans strain SC5314 in mice.

Detailed ways

The present invention will be further described in detail below in conjunction with the embodiments and the accompanying drawings, but the embodiments of the present invention are not limited thereto.

Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.

Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

Example 1 Detection of kahweol and kahweol antibacterial activity

1. Test method:

(1) Activation of Candida albicans strains:

Candida albicans standard strain SC5314 (named also ATCC MYA-2876) was activated in LB solid medium (tryptone 10g/L, yeast extract 5g/L, NaCl 10g/L, agar 15g/L), placed in 30 Cultivate overnight in an incubator.

(2) Effects of cafestol and cafestol on mycelia of Candida albicans strain SC5314:

Pick the SC5314 strain on the LB solid plate (tryptone 10g/L, yeast extract 5g/L, NaCl 10g/L, agar 15g/L) and inoculate it in GMM culture medium (6.7g/L YNB, 0.2% glucose) medium, 30°C, 200rpm shaking culture overnight, measure the OD ₆₀₀ of the bacterial solution, and dilute the bacterial solution to OD ₆₀₀ =0.1 with GMM. Take 500 μL of bacterial liquid in a 1.5mL EP tube, add kahweol and kahweol, the final concentration is 30 μg/mL; set DMSO control at the same time; in the DMSO control group, DMSO is added to the obtained from bacterial fluid. Shake and mix well, and incubate in a water bath at 37°C. After 6 hours, centrifuge at 5,000 rpm for 10 minutes, discard the supernatant, add 40 μL of GMM culture medium to resuspend the bacteria, and observe the formation of hyphae under a Zeiss Axioplan 2 microscope, taking different fields of view Photographs were taken, and the mycelium formation rate was calculated.

(3) Effects of cafestol and cafestyl alcohol on the adhesion of Candida albicans strain SC5314:

(a) Recovery and cultivation of human non-small cell lung cancer cell line A549 cells: transfer the frozen-thawed A549 cells to DMEM medium (Gibco Company) containing 10% (v/v) FBS, 37 ° C, 5% CO ₂ conditions overnight.

(b) A549 cell preparation: A549 cells were cultured overnight in a 96-well plate at a cell concentration of 0.5×10 ³ cells/well in high-glucose medium DMEM containing 10% fetal bovine serum. When the cells covered 80% of the bottom of the 96-well plate, the culture medium was discarded, and the cells were washed 3 times with 1×PBS.

(c) Pick the SC5314 strain on the LB solid plate, inoculate it in GMM culture solution (6.7g/L YNB, 0.2% glucose), culture it overnight at 30°C with shaking at 200rpm, and measure the OD ₆₀₀ of the bacterial solution. Then adjust and dilute the bacterial solution to OD ₆₀₀ =0.5 with cell maintenance solution (DMEM containing 1% v/v FBS). Dilute the cafestyl alcohol mother liquor and the cafestyl alcohol mother liquor (the mother liquor is prepared with DMSO) with DMSO to dilute the concentration of 6mg/mL, 3mg/mL, 1.5mg/mL, 0.75mg/mL respectively, and then the cafestol alcohol The diluent and the diluent of cafestyl alcohol were respectively mixed with the cell maintenance solution containing bacteria at a volume ratio of 1:199, oscillated and mixed to obtain the test solution A, and the final concentrations of cafestyl alcohol and cafestyl alcohol in the test solution A were respectively 30μg/mL, 15μg/mL, 7.5μg/mL, 3.75μg/mL. Add 100 μL/well to the 96-well plate of the cultured cells in step (b), and set 4 repetitions for each treatment; set up the treatment of adding only DMSO at the same time, wherein, the volume ratio of DMSO and bacteria-containing cell maintenance solution is 1: 199 ratio mixed to obtain the test solution B. The 96-well plate was incubated at 37° C., and the culture medium was discarded after 1.5 h, and 100 μL of crystal violet solution with a concentration of 0.5% (w/v) was added to each well for 45 min at room temperature. Discard the crystal violet, wash with ice ddH ₂ O 10 times, add 100 μL of 75% by volume ethanol solution, leave at room temperature for 30 min, measure OD ₅₇₀ , and process data with GraphPad Prism 6 software.

(4) Effects of cafestol and cafestyl alcohol on biofilm formation of Candida albicans strain SC5314:

The SC5314 strain on the LB solid plate was picked, inoculated in GMM culture solution (6.7g/L YNB, 0.2% glucose), cultured overnight at 30°C with shaking at 200rpm, and the OD ₆₀₀ of the bacterial solution was measured. Then the bacterial solution was diluted to OD ₆₀₀ =0.1 with GMM culture solution to obtain the GMM culture solution containing bacteria. Dilute the cafestyl alcohol mother liquor and the cafestyl alcohol mother liquor with DMSO to dilute the concentration of 6mg/mL, 3mg/mL, 1.5mg/mL, 0.75mg/mL, and then dilute the cafestyl alcohol dilution and cafeteria alcohol solution was mixed with the GMM culture solution containing bacteria at a volume ratio of 1:199, oscillated and mixed to obtain test solution D, and the final concentrations of cafestol and cafestyl alcohol in test solution D were 30 μg/mL and 15 μg/mL, respectively. mL, 7.5 μg/mL, 3.75 μg/mL. 100 μL/well was added to a 96-well plate, and 4 replicates were set for each treatment, and a control group with only DMSO was set at the same time. The 96-well plate was incubated at 37° C., and the culture medium was discarded after 8 hours, and 100 μL of crystal violet with a concentration of 0.5% w/v was added for 45 minutes at room temperature. Discard the crystal violet, wash with ice ddH ₂ O 10 times, add 100 μL of 75% ethanol, let stand at room temperature for 30 min, measure OD ₅₇₀ , and process data with GraphPad Prism 6 software.

(5) Determination of the influence of cafestol and cafestyl alcohol on the growth of Candida albicans strain SC5314:

Pick a single colony of strain SC5314 and inoculate it in GMM medium (6.7g/L YNB, 0.2% glucose), culture overnight at 30°C with shaking at 200rpm, measure the OD ₆₀₀ of the bacterial solution, and dilute the bacterial solution to OD ₆₀₀ =0.05 with GMM. Dilute the cafestyl alcohol mother liquor and the cafestyl alcohol mother liquor with DMSO to dilute the concentration of 6mg/mL, 3mg/mL, 1.5mg/mL, 0.75mg/mL, and then dilute the cafestyl alcohol dilution and cafeteria alcohol solution was mixed with the GMM culture medium containing bacteria at a volume ratio of 1:199, and added to a 100-well plate at an amount of 300 μL/well. Each treatment was set up with 3 replicates, and at the same time, only DMSO was added to the treatment. Place in a growth curve analyzer at 30°C and 200 rpm, measure the OD ₆₀₀ value every 2 hours, observe the experimental results after 20 hours, and process the data with GraphPad Prism 6.

(6) Effects of cafestol and cafestol on the cytotoxicity of Candida albicans strain SC5314:

(b) A549 cell preparation: A549 cells were cultured overnight in a 96-well plate at a cell concentration of 1.5×10 ⁴ cells/well in high-glucose medium DMEM containing 10% fetal bovine serum. When the cells covered 80% 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: pick fresh SC5314 and inoculate it in GMM culture medium (6.7g/L YNB, 0.2% glucose), shake and cultivate overnight at 30°C and 200rpm; use cell maintenance medium (containing 1% FBS DMEM) adjusted to OD ₆₀₀ =1.0, and then diluted 10 times with cell maintenance solution (≈10 ⁸ CFU/mL) to obtain cell maintenance solution containing bacteria.

(d) Cytotoxicity assay:

a) Determination of the cytotoxic effect of kahweol and kahweol itself on cells: Dilute kahweol and kahweol to 6 mg/mL with DMSO, respectively, and add them to the cell maintenance solution. The final concentrations of kahweol and kahweol are 30 μg, respectively. /mL to obtain test solution E; at the same time, set up a control group and take the same volume of DMSO to obtain test solution F. Add test solution E and test solution F to the prepared A549 cells at 100 μL/well, place them in a 37°C, 5% CO ₂ cell incubator and incubate them for 8 hours, with 4 replicates per treatment.

b) Determination of the cytotoxicity of kahweol and kahweol to Candida albicans: Dilute kahweol and kahweol with DMSO to concentrations of 6 mg/mL, 3 mg/mL, 1.5 mg/mL, and 0.75 mg/mL, respectively. mL of the diluted drug solution, and then the cafestyl alcohol dilution and the cafestyl alcohol dilution were mixed with the cell maintenance solution containing bacteria at a volume ratio of 1:199 to obtain the test solution G. The final concentrations in solution G were 30 μg/mL, 15 μg/mL, 7.5 μg/mL, and 3.75 μg/mL respectively. In test solution G, the volume content of DMSO was basically the same; sulfone) as a control, wherein DMSO and bacteria-containing cell maintenance solution were mixed in a volume ratio of 1:199 to obtain test solution H. Add test solutions G and H to the prepared A549 cells at 100 μL/well, respectively, and place them in a 37°C, 5% CO ₂ cell incubator for 8 hours, with 4 replicates per treatment.

c) Refer to Promega CytoTox

The NonRadioactive Cytotoxicity Assay method was used to measure cellular LDH activity, and then the data was processed with GraphPad Prism 6.

(7) Determination of the influence of cafestol and cafestyl alcohol on the oral infection of Candida albicans strain SC5314 mice:

(a) Breeding of mice: 6-8 week-old male BALB/c mice purchased from Guangdong Experimental Animal Center were bred in the Experimental Animal Center of South China Agricultural University. Randomly assign 8 mice/group, weigh and record and mark each mouse.

(b) Preparation of Candida albicans suspension: activate Candida albicans standard strain SC5314 in LB solid medium (tryptone 10g/L, yeast extract 5g/L, NaCl 10g/L, agar 15g/L), namely Place in a 30°C incubator overnight. Take a single colony and inoculate it in GMM culture medium (6.7g/L YNB, 0.2% glucose), and cultivate overnight at 30°C on a shaker at 200rpm. Centrifuge at 4°C and 3000rpm for 5min, wash twice with pre-cooled sterile PBS, count by blood cell counting method, and prepare a bacterial suspension with a concentration of 3×10 ⁶ CFU/mL. Take 5mL of bacterial suspension for later use. Preparation Take 5 mL of PBS and add 25 μL of kahweol diluent (concentration: 6 mg/mL, solute is DMSO) to the final concentration of kahweol to 30 μg/mL, take 5 mL of PBS, add 25 μL of DMSO to it.

(c) Mice oral infection experiment: the mice were weighed one day before infection and each injected with 0.1mL/10g of hydrocortisone (22.5mg/mL, before inoculation, the mice were anesthetized with 10% chloral hydrate , the anesthetized mice were placed flat in a constant temperature environment of 37°C, soaked in the bacterial suspension with sterile cotton balls for 3 minutes and then placed under the tongues of the mice for 90 minutes; at the same time, a blank control group was set up, and the blank control group soaked the sterile balls with PBS solution Put it under the tongue of the mouse for 90 minutes after cotton. Then, on the 1st, 3rd, and 5th day, each mouse was fed with 100 μL/10 g of cafeteria alcohol dilution (the concentration was 30 μg/mL, and the solute was DMSO); at the same time, a positive control group was set , the positive control group was gavaged with PBS containing DMSO. On the 6th day, the mice were sacrificed, and the tongues of the mice were dissected and further observed in pathological sections.

2. Experimental results

(1) Caffeol and cafestol inhibit the mycelium formation of Candida albicans strain SC5314

Figure 1 is a photograph of the mycelium formation of Candida albicans observed under a microscope, in which the same volume of DMSO was added to the bacterial solution according to the kahwegol and kahweol treatment groups, and cultured at 37°C as a control group to exclude the effect of DMSO on white Effects on Candida hyphae formation. Calculate the inhibition rate of mycelium formation by photos: take 3 photos (3 photos with 3 repeated values) for one sample, count the mycelia on the photos, and the mycelium formed with DMSO is 100%. The results are shown in Figure 2, kahweol and cafestyl alcohol have obvious mycelial inhibitory effect on Candida albicans SC5314 respectively, and after treatment with kahweol or kahweol at a final concentration of 30 μg/mL, Candida albicans basically has no Mycelium formation.

(2) Caffeol and cafestol inhibit the adhesion of Candida albicans strain SC5314

As shown in FIG. 3 , taking DMSO as a reference, the adhesion of Candida albicans on polystyrene after being treated with kahweol and kahweol at a final concentration of 30 μg/mL was reduced by more than 50%. It shows that kahweol and cafestyl alcohol have a certain inhibitory effect on the adhesion of Candida albicans SC5314 respectively.

(3) Effects of cafestol and cafestyl alcohol on biofilm formation of Candida albicans strain SC5314:

As shown in FIG. 4 , taking DMSO as a reference, the biofilm formation of Candida albicans on polystyrene after being treated with cafestol or cafestyl alcohol (30 μg/mL) was reduced by more than 90%. It shows that cafestol or cafestyl alcohol has a good inhibitory effect on the biofilm formation of Candida albicans SC5314.

(4) High concentration of kahweol has a slight inhibitory effect on the cell growth of Candida albicans strain SC5314, and low concentration of kahweol and kahweol have no inhibitory effect;

The results showed that using DMSO as a control, cafestyl alcohol had a slight inhibitory effect on the growth of Candida albicans strain SC5314 at a concentration of 30 μg/mL ( FIG. 5A ), but cafestyl alcohol had no inhibitory effect ( FIG. 5B ). This result indicates that the therapeutic effect of cafestol or cafestyl alcohol on Candida albicans strain SC5314 is not mainly achieved by killing bacterial cells, and thus drug resistance is not easy to develop.

(5) Caffeol or cafestol has a good inhibitory effect on the cytotoxicity of Candida albicans strain SC5314

We detect the cytotoxicity by detecting the release amount of LDH. When detecting the cytotoxicity of Candida albicans, we take the LDH release amount of the DMSO group added to Candida albicans SC5314 as 100%, and thereby standardize other LDH release ratio of kahweol or kahweol group. The results are shown in Figure 6, the data shown are the average of 4 biological replicates, and the error bars reflect the standard deviation.

The results of the cytotoxicity experiment showed that, using DMSO as a control, kahwegol or cafestyl alcohol had no toxicity to cells at a concentration of 30 μg/mL in the absence of Candida albicans, as shown in FIG. 6A .

And under the condition of adding Candida albicans SC5314, using DMSO as a control, Figure 6B shows that cafestol or cafestyl alcohol has a good protective effect on inhibiting the invasion of cells by SC5314; /mL concentration, the virulence of Candida albicans was reduced to below 30%.

(6) Caffeol has a good inhibitory effect on the oral infection of Candida albicans strain SC5314 in mice

By observing the formation of Candida albicans on the tongues of mice, we found that the tongues of the mice in the Candida albicans control group formed a thick mycelial layer of Candida albicans, while in the PBS blank control group and kahweol treatment group, the small There was no mycelium formation in the mouse tongue; pathological sections (as shown in Figure 7) showed that there was a mycelial layer of Candida albicans on the upper layer of the mouse tongue cells in the Candida albicans control group, but there was no mycelial layer in the PBS blank control group and the kahweol-treated group.

The above-mentioned embodiment is a preferred embodiment of the present invention, but the embodiment of the present invention is not limited by the above-mentioned embodiment, and any other changes, modifications, substitutions, combinations, Simplifications should be equivalent replacement methods, and all are included in the protection scope of the present invention.

Claims

Application of cafestol or derivatives thereof in preparation of anti-candida medicament or anti-candida daily necessities.
The application of cafestol or its derivatives according to claim 1 in the preparation of anti-Candida albicans medicine or anti-Candida albicans daily necessities, is characterized in that: described anti-Candida albicans medicine has the mucoid that inhibits Candida albicans Adhesion, mycelia formation, biofilm formation, pathogenic drug.
The application of cafestol or its derivatives according to claim 1 in the preparation of anti-Candida albicans medicaments or anti-Candida albicans daily necessities, characterized in that: the anti-Candida albicans medicaments are for prevention and/or treatment The medicine for Candida albicans infection, and one or both of the medicines for preventing and/or treating infectious diseases caused by Candida albicans.
The use of cafestol or its derivatives according to claim 1 in the preparation of anti-Candida albicans medicine or anti-Candida albicans daily necessities, characterized in that: said daily necessities are mouthwash or vaginal lotion.