CN114010790B

CN114010790B - Use of fluorescent whitening agents against fungal infections

Info

Publication number: CN114010790B
Application number: CN202111346628.3A
Authority: CN
Inventors: 王万能; 成福
Original assignee: Chongqing University of Technology
Current assignee: Chongqing University of Technology
Priority date: 2021-11-15
Filing date: 2021-11-15
Publication date: 2023-06-02
Anticipated expiration: 2041-11-15
Also published as: CN114010790A

Abstract

The application of the stilbene fluorescent whitening agent in inhibiting the activity of fungi provided by the invention has excellent antibacterial activity against dermatophytes such as trichophyton rubrum and microsporona gypseum, and also has antibacterial effect against drug-resistant candida albicans. In addition, the invention provides a fluorescent whitening agent 85/linalool gel preparation which is stable, has wide temperature application range and has no skin irritation and toxicity. The preparation is used for treating a guinea pig model infected by trichophyton rubrum, can control the occurrence and development of skin tinea, has an antibacterial effect better than that of a positive drug terbinafine in a treatment period, and is a potential stable high-efficiency antifungal drug.

Description

Use of fluorescent whitening agents against fungal infections

Technical Field

The invention belongs to the field of biological medicine, and particularly relates to an application of a fluorescent whitening agent in inhibiting fungal activity, in particular to an application in inhibiting dermatophytosis caused by trichophytosis rubrum infection.

Background

Superficial fungal infections are dermatophytes caused by keratiphilic fungal infections, and are commonly seen as tinea corporis, tinea pedis, tinea cruris, onychomycosis, tinea capitis, tinea facialis, etc., affecting about 20% -25% of the population. Pathogenic fungi that cause fungal infections in humans are reported to fall into three main categories: trichophyton (Trichophyton) and epidemic rhodophyton (epidermophyta) and microsporoum (microsporophyta), wherein trichophyta rubrum (Trichophyton rubrum) is the main pathogenic bacteria causing dermatomycosis, accounting for more than 60% of human dermatomycosis. At present, fungal skin diseases caused by superficial fungi always afflict normal life of people, and the fungal skin diseases often have the characteristics of long treatment course, difficult cure, easy recurrence, easy infection and the like, which bring pain and trouble to patients and families of the patients. In addition, with the development of medicine, the wide application of broad-spectrum antibiotics, corticosteroids and immunosuppressants, and changes in the lifestyle of people, the incidence of dermatomycosis increases year by year, and the aged, diabetic and immunocompromised people are more susceptible to dermatomycosis. It is worth noting that the prior antifungal drug treatment has obvious drug resistance, and the traditional oral drug has great hepatotoxicity and renal toxicity and has a certain degree of injury to human bodies. There is a need for safer, more effective antimicrobial therapies and alternative therapies to alleviate the problem of pathogenic resistance. However, in recent years, the development of new antifungal drugs is delayed, the ever-increasing drug demands are difficult to meet, and the discovery and development of a novel external antifungal drug is of great significance due to the specificity of dermatomycosis.

Fluorescent brightening agent 85 (Fluorescent Brightener, abbreviated as FB 85, trade name: fluorescent brightening agent VBL) is the dominant product of stilbene fluorescent brightening agent, the academic name 4,4 '-bis- (4-light ethylamino-anilino-1, 3, 5-triazin-2-yl) amino-stilbene 2,2' -disulfonic acid sodium salt is widely used in pH chemical sensing materials, chemical sensors, light emitting diodes, biological staining and other aspects, has been the market for more than 50 years so far, is the fluorescent brightening agent variety with the largest yield and the largest application in China at present, and the consumption amount accounts for more than 85% of the fluorescent brightening agent used in the Chinese detergent industry, and the fluorescent brightening agent has no relevant report in antifungal aspect as a medicament.

Disclosure of Invention

In view of the above, it is an object of the present invention to provide an application of fluorescent whitening agent in inhibiting fungal activity, it is a second object of the present invention to provide a gel preparation for resisting fungal infection, and it is a third object of the present invention to provide an application of the gel preparation for resisting fungal infection in treating dermatologic diseases of fungal infection.

Technical proposal

In order to achieve the above purpose, the present invention provides the following technical solutions:

1. the use of an optical brightening agent in inhibiting fungal activity, said optical brightening agent being a stilbene optical brightening agent.

As one of the preferred technical features, the stilbene-based fluorescent whitening agent includes, but is not limited to, fluorescent whitening agent 85, fluorescent whitening agent 71, fluorescent whitening agent 113, fluorescent whitening agent 134, fluorescent whitening agent 210, fluorescent whitening agent 220, fluorescent whitening agent CFW or fluorescent whitening agent ryuux BSU.

As one of the preferred technical features, the fungus includes, but is not limited to, trichophyton rubrum or microsporum gypsilosis.

As one of the preferable technical features, the fungus is a drug-resistant fungus.

As one of the preferable technical characteristics, the drug-resistant fungus is drug-resistant candida albicans.

2. A gel formulation for combating a fungal infection, said gel formulation comprising: fluorescent whitening agent 85, linalool, glycerol, propylene glycol, triethanolamine, carbomer 940 and ethylparaben.

As one of the preferred technical features, the formulation components include: fluorescent brightening agent 851-3 mg/mL, linalool 0.5-1 mg/mL, glycerol 20-80 mg/mL, propylene glycol 2-8 mg/mL, triethanolamine 10-12 mg/mL, carbomer 9404-10 mg/mL and ethylparaben 0.2-0.4 mg/mL.

3. The use of said gel formulation against fungal infections for the treatment of dermatological disorders involving fungal infections.

As one of the preferred technical features, the fungus is trichophyton rubrum.

The beneficial effects are that:

the stilbene fluorescent whitening agent overcomes the defect that the existing antifungal drug can only inhibit the action of new cells, has high affinity with B glycosidic bond in the cell wall of fungi, can destroy the stress of the cell wall, radically kills fungi by interfering the normal synthesis and assembly of glucan, has no cell wall in mammalian cells, and has good targeting. Furthermore, most biosynthetic enzymes and cell wall components in the cell wall are unique to fungi and drugs targeting the cell wall are ideal targets for the specific location of the fungal cell wall and the reason that the cell wall primarily mediates fungal-host interactions. In addition, the research also finds that the fluorescent whitening agent 85 has better inhibition effect on drug-resistant strain candida albicans, which reveals the unique antifungal effect of the stilbene fluorescent whitening agent 85, and is hopeful to be used for serious fungal infection and reduce the occurrence of drug resistance. The linalool provided by the invention can act on the cell membrane of trichophyton rubrum, can change the permeability of the cell membrane, can play a role in inhibiting the synthesis of ergosterol which is a cell membrane component of trichophyton rubrum, can be used in combination with the fluorescent whitening agent 85, and can play a role in inhibiting trichophyton rubrum in various ways, so that an excellent antibacterial treatment effect is realized.

Drawings

Fig. 1: the inhibition of fungi by fluorescent whitening agent 85 is shown. A is Trichophyton rubrum MIC ₈ 0 determination result, B is MIC of Microsporum gypseum ₈₀ The measurement result is C is the measurement result of trichophyton rubrum MFC, D is the measurement result of Microsporum gypseum MFC, E is the antibacterial activity of Trichophyton rubrum, and F is the antibacterial activity of Microsporum gypseum.

Fig. 2: fluorescent whitening agent 85 inhibits drug-resistant candida albicans effect pattern. A is the antibacterial activity of fluorescent whitening agent 85 on drug-resistant candida albicans, a is a flat-plate front view, B is a flat-plate back view, and B is the MIC of fluorescent whitening agent 85 for resisting drug-resistant candida albicans ₈₀ And C is the measurement result of fluorescent whitening agent 85 and drug resistant candida albicans MFC.

Fig. 3: fluorescent whitening agent 85 was used to treat trichophyton rubrum. A is the effect of fluorescent whitening agent 85 on dextran of cell wall components of trichophyton rubrum, B is the microstructure observed by a scanning electron microscope after the fluorescent whitening agent 85 is acted on trichophyton rubrum, a, B and c are the results observed by a control group at 5000x, 20000x and 50000x, and d, e and f are the results observed by an experimental group at 5000x, 20000x and 50000x, respectively.

Fig. 4: the appearance state of the prepared gel. A is the appearance state of the prepared gel, B is the research result of the heat and cold resistance of the prepared gel, a and B are respectively the front and back diagrams of a constant temperature drying heat resistance experiment at 60 ℃, C and d are respectively the front and back diagrams of a freezing cold resistance experiment at-20 ℃, C is the measurement result of the stability of the prepared gel, a is the gel state before centrifugation, and B is the state after gel centrifugation.

Fig. 5: gel formulations observed for 1-48h were tested for skin irritation in SD rats.

Fig. 6: a graph was constructed for a trichophyton rubrum infected guinea pig model. A is the case of back skin abrasion of guinea pigs, B is the case of 2D after inoculation of the guinea pigs with trichophyton rubrum, C is the case of 7D after infection of the guinea pigs with trichophyton rubrum, D is the case of observing hyphae in dander at the infection site under an optical microscope, E, F is the positive and negative image of dander inoculation culture at the infection site of the guinea pigs in SDA medium.

Fig. 7: blank, positive, experimental, model groups were treated for skin changes 1d before, 1d after, 5d after, and 10d after the treatment.

Fig. 8: animal score plots for blank, positive, experimental, model.

Detailed Description

The present invention will be described in further detail below in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific examples described herein are for illustrative purposes only and that the following experiments demonstrate the efficacy of the optical brightening agent 85 against dermatophytes and the in vitro efficacy of the prepared optical brightening agent 85 against dermatophytes, as well as other advantages and efficacy of the present invention as will be readily apparent to those skilled in the art from the disclosure herein. The invention may be embodied or practiced in other different specific embodiments and is not limited to the specific details. The details in the description may be modified or varied from different points of view and applications without departing from the spirit of the invention.

Example 1

Fluorescent whitening agent 85 inhibits fungal activity

Preparing fluorescent whitening agent 85 with the final concentration of 0.3125 mg/mL-10 mg/mL, sterilizing and filtering for later use. Spore suspension was prepared by adding sterile physiological saline to plates after 7d of culture on glucose agar (SDA) and washing with Trichophyton rubrum spores. And (3) uniformly coating an appropriate amount of spore suspension on an SDA flat plate, dividing the culture medium into three areas such as an experimental part and a control part after the surface of the culture medium is completely dried, punching by using an 8mm puncher, adding an optical brightening agent 85 with the concentration of preferably 2.5mg/mL into the holes, and incubating for 5-7d in a 30 ℃ incubator for observation. As a result, as shown in FIG. 1 at E, F, the fluorescent whitening agent 85 exhibited strong sensitivity and antibacterial activity against both strains of Trichophyton rubrum and Microsporum gypseum as test strains.

In order to study the potential of the fluorescent whitening agent 85 as an antifungal agent for skin, the antifungal activity of the fluorescent whitening agent 85 against trichophyton rubrum is further determined, the antifungal activity of the fluorescent whitening agent 85 against trichophyton rubrum is studied by a microdilution method with reference to a CLSI M38-A2 antifungal drug sensitivity test method, and the inhibition of the fluorescent whitening agent 85 at different concentrations against trichophyton rubrum is statistically evaluated. The Minimum Inhibitory Concentration (MIC) and minimum bactericidal concentration (MFC) of the fluorescent whitening agent 85 against trichophyton rubrum, microsporum gypseum, are shown in fig. 1 a-D. The results showed that with increasing drug concentration, the lower the growth rate of trichophyton rubrum and microsporum gypseum, the more turbid the growth of trichophyton rubrum and microsporum gypseum was observed in the corresponding drug-sensitive plate wells at fluorescent whitening agent 85 concentrations of 2.4 μg/mL, 4.9 μg/mL and 9.8 μg/mL, respectively. When the drug concentration was increased, the growth of trichophyton rubrum and microsporopsis gypseum had been completely inhibited (100%), and their bactericidal effect was confirmed by transfer experiments, and it was determined that the MFC of fluorescent whitening agent 85 against trichophyton rubrum and microsporopsis gypseum was 19.5 μg/mL.

Example 2

Inhibition of drug-resistant bacteria by fluorescent whitening agent 85

The fluorescent whitening agent 85, which was measured as in example 1, had an antibacterial effect against drug-resistant candida albicans (deposited at the center of the university of Chongqing pharmaceutical and bioengineering laboratory), as shown in fig. 2 a (a) to b). As the concentration of the drug increases, the inhibition effect thereof increases, and the MIC of the fluorescent whitening agent 85 for the drug-resistant candida albicans is measured ₈₀ 113.28 μg/mL as shown in FIG. 2B.

Fluorescent whitening agent 85 was assayed for drug resistant candida albicans MFC. A YPD plate was coated with 78.1. Mu.g/mL of fluorescent whitening agent 85, 156.3. Mu.g/mL, 312.5. Mu.g/mL of the drug-resistant Candida albicans strain treated with the fluorescent whitening agent 85 solution, and the results are shown in FIG. 2C. The results showed a large amount of strain growth at a concentration of 78.1. Mu.g/mL of fluorescent whitening agent 85, indicating that fluorescent whitening agent 85 was not effective against drug-resistant Candida albicans at this concentration, and further, we found that when we measured MFC at 156.3. Mu.g/mL of fluorescent whitening agent 85, the results showed that there was still some bacterial growth, whereas when 312.5. Mu.g/mL of fluorescent whitening agent 85 was treated, the plates showed aseptic growth, and therefore the MFC value of fluorescent whitening agent 85 against drug-resistant Candida albicans was 312.5. Mu.g/mL.

Example 3

Antifungal mechanism of fluorescent whitening agent 85

The effect of the fluorescent whitening agent 85 on the dextran component of the cell wall of trichophyton rubrum was measured by infrared spectrometry, and the change of the cell wall of trichophyton rubrum after the treatment of the fluorescent whitening agent 85 was observed by using a scanning electron microscope. After the trichophyton rubrum is treated by the fluorescent whitening agent 85, the thalli are collected, washed with water and freeze-dried. The dry cells were suspended in 50ml of 1 mol/L NaOH solution and reacted at 100℃for 1 hour. Centrifuging at 6000r/min for 15min, discarding supernatant, suspending the precipitate with 50mL of 0.75mol/L NaOH solution, treating at 100deg.C for 15min, cooling, adjusting pH to 4.5 with HCI, washing with water for 2 times, dehydrating with absolute ethanol, adding 20mL diethyl ether for dehydration, air drying, tabletting with KBr, and making red light spectrum.

As shown in fig. 3 a, the characteristic absorbance peak-to-peak values of the treated strain β -glycosidic bond, C-O, C-H, -OH, c=o were reduced compared to the control strain; indicating that the beta-glycosidic bond and the C-O bond, -OH, c=o of the glucan are destroyed after treatment with the fluorescent whitening agent 85. The results show that the intermolecular glycosidic bond of the glucan monosaccharide is destroyed and inhibited, and the normal synthesis and assembly of the cell wall are hindered, so that the growth of fungi is inhibited. In addition, as shown in (a) to (f) of FIG. 3, scanning electron microscopy found that after the drug treatment, trichophyton rubrum spores were difficult to develop into hyphae, and fluorescent whitening agent 85 inhibited germination of trichophyton rubrum spores. In addition, the figure also shows that most of spores have surface wrinkles, blurred contours, depressions on the cell surface, reduced integrity of the cell wall and obvious changes in morphology, and the result further proves that the fluorescent whitening agent 85 can act on the cell wall of trichophyton rubrum spores, thereby exerting antifungal effect.

Example 4

Preparation of fluorescent whitening agent 85/linalool gel preparation

Formula 1:

optical brightening agent 851mg/mL, linalool 0.5mg/mL, glycerol 20mg/mL, propylene glycol 2mg/mL, triethanolamine 10mg/mL, carbomer 9404mg/mL, ethylparaben 0.2mg/mL.

Formula 2:

fluorescent whitening agent 853mg/mL, linalool 1mg/mL, glycerol 80mg/mL, propylene glycol 8mg/mL, triethanolamine 12mg/mL, carbomer 94010mg/mL, ethylparaben 0.4mg/mL.

Taking 50g of the gel preparation of formula 1 as an example, the preparation method is as follows:

(1) Slowly adding 0.2g carbomer 940 into a beaker containing sterile water, stirring, sealing the mouth of the beaker with a preservative film after the carbomer is completely immersed in the water, and placing in a refrigerator at 4 ℃ for 24 hours or at normal temperature for 4 hours to fully swell the carbomer.

(2) 10mg of ethylparaben, 1g of glycerol, 0.1g of propylene glycol and 5mL of sterile water are weighed, a centrifuge tube is placed on a vortex mixer for full dissolution, the solution after uniform mixing is filtered by a filter membrane of 0.22 mu m. 8550mg of fluorescent whitening agent is accurately weighed and dissolved in a proper amount of water, and a filter membrane of 0.22 mu m is filtered and protected from light for standby.

(3) Mixing fully swelled carbomer with the weighed ethylparaben, glycerol and propylene glycol, and stirring uniformly.

(4) Adding the fluorescent whitening agent 85 solution into the gel in the step (3), fully mixing, adding 10mg/mL of triethanolamine, and finally adding sterile water to make up to 50g. After the gel preparation is prepared, the preservative film is sealed and placed in a refrigerator at 4 ℃ for 24 hours, so that the medicine inside the gel preparation is fully diffused, and the preparation is more uniform.

Example 5

Stability and temperature sensitivity performance study of fluorescent whitening agent 85/linalool gel preparation

An optical brightener 85/linalool gel formulation was prepared according to the preparation method described in example 4, in the form shown in figure 4 a. 15g of a fluorescent whitening agent 85/linalool gel preparation sample is taken and placed in a centrifuge tube, and after centrifugation for 30min at 2500r/min, whether water is separated out and layering phenomenon is observed. As shown in FIG. 4C (a, b), the prepared carbomer gel showed no exudation of moisture after centrifugation, and the gel composition was uniform without delamination, and the results indicated that the carbomer gel under this formulation was stable. In addition, the sample is put into a constant temperature drying oven to be dried for 6 hours at 60 ℃, taken out, and put into room temperature to observe whether the appearance changes or not, and whether gel has layering or not is observed; and placing the sample in a refrigerator at the temperature of minus 20 ℃ for 24 hours, taking out and thawing, and observing whether the appearance changes or not and whether gel has layering or not. As shown in B (a-d) in FIG. 4, after 6 hours of drying, the gel still has no obvious change, and no layering and liquid seepage phenomena, which indicates that the gel has thermal stability. In addition, after 24 hours of freezing, the gel has no phenomenon of layering and liquid seepage, which indicates that the gel has low-temperature tolerance.

Example 6

Fluorescent whitening agent 85/linalool gel formulation for treating dermatologic diseases with fungal infections

(1) The fluorescent whitening agent 85 gel preparation has no irritation to skin

4 healthy adult male SD Rat animals are selected and kept in a single cage, and the animals are adapted to at least 3d in the test environment before the test. The feed is fed by conventional feed and can be drunk freely. Untreated skin on the opposite side of each animal served as a control. And (3) cutting or shaving hairs on two sides of the spine of the experimental animal 24 hours before the experiment, wherein the hairs can not damage epidermis, and selecting the animal with intact skin health after about 3cm multiplied by 3cm and 24 hours of the hair removal range. A2.5 mg/mL sample of 0.5g was uniformly applied to the right skin of the rat, with the left side as a control. Covered with 4 layers of gauze (2.5 cm. Times.2.5 cm) and secured with non-irritating adhesive tape and bandages. The application time was 4h using the blocking test. After the test, the residual test sample was removed with warm water. Skin reactions at the test sites were observed at 1h, 24h, 48h and 72h after the test samples were removed, skin reactions were scored according to table 1, comprehensive evaluation was performed on the average of the integral of the test animals, and skin irritation intensities were determined according to table 2 from the highest integral average at each observation time point of 24h, 48h and 72 h. The results are shown in Table 3, with an integrated mean of 0, indicating no skin irritation. The back skin of the experimental rat shown in fig. 5 has no obvious change compared with the control area after the treatment of the medicine, and the gel has no irritation to skin tissues, thus laying a foundation for the clinical application of the gel.

TABLE 1 skin irritation test animal scoring criteria

Table 2 skin irritation test animal score

TABLE 3 skin irritation test animal scoring results

(2) Fluorescent whitening agent 85 gel composition for treating dermatologic diseases infected with trichophyton rubrum

24 healthy guinea pigs of male and female halves, weighing about 300g, were randomly divided into 4 groups, a blank control group, a model control group, a positive control group and an experimental group. All experimental animals were given 1 triamcinolone acetonide daily at 20mg/kg and injected intramuscularly at the thigh 3d prior to inoculation. Adjusting the concentration of trichophyton rubrum suspension to 1×10 ⁸ CFU/mL, the back skin of guinea pigs is abraded with sterilized coarse sand paper after depilating treatment of 2.5cm×2.5cm on the back of guinea pigs before inoculation until the skin appears as blood seepage. Except for the blank group, the other groups are not inoculated with trichophyton rubrum, and simultaneously the dosage of the dexamethasone injection is 7.5mg/kg, and the injection is carried out once every other day and is continuously carried out for 4 days. And 4d after inoculation, scraping a little dander at the back of the animal for normal microscopic examination of 10% potassium hydroxide, and performing once every other day until the microscopic examination is observed to generate hyphae spores, so that the animal model is proved to be successfully constructed.

After the guinea pig infection model is successfully constructed (as shown in A-F in figure 6), terbinafine hydrochloride gel is smeared on the positive control group, and the dosage is 2.0mg/cm ² Each time in the morning and evening; the experimental group is smeared with the prepared gel preparation containing 2.5mg/mL fluorescent whitening agent 85 with the dosage of 2.0mg/cm ² Each time in the morning and evening. The blank control group and the model control group do not need to be treated by medicines, and the same amount of sterile physiological saline is used for the treatmentInstead of. The positive control group, the experimental group and the model group were scraped every other day after administration, and the skin and hair growth of each group of guinea pigs was observed by microscopic examination of the dander at the place where the guinea pigs were inoculated, and the skin loss disappearance time of each guinea pig was recorded and the recovery of the skin damage was scored for 1d before administration, 1d after administration, 5d after administration and 10d after administration of the guinea pigs, with the scoring criteria as shown in table 4.

TABLE 4 skin score evaluation criteria for guinea pigs

As a result, as shown in FIG. 7, in the control group guinea pigs not infected with Trichophyton rubrum, hair growth was relatively good, hair removal was smooth and flat, no sign of skin injury was observed, and skin diseases of the infected guinea pigs were severely damaged, after the Trichophyton rubrum infection of the back epidermis tissue of the guinea pigs, the skin was vasodilated and edematous, accompanied by hyperkeratosis and hypokeratosis of the skin tissue, and the like, at 4d of infection, the degree of edema and skin injury were slightly decreased. After animal molding is successful, the treatment is started by smearing the medicine within 48 hours, and the compound gel latex containing the fluorescent whitening agent 85 and linalool is administered to an experimental group once a day, wherein the dosage of each time is 0.2g/cm ² . The dosage of the positive control terbinafine hydrochloride emulsifiable paste group is 0.2g/cm ² Once daily. After 1d of treatment with the drug, the skin tissues of the back of the guinea pigs in the experimental group and the positive control group had reduced redness and swelling, and had dandruff dropped, and hair was gradually grown. After 5d of the drug, after 10d of the drug treatment, the skin of the lesion part of the experimental group is restored to be smooth, the result of the mycoscopic examination is negative, the hair grows normally, and no dandruff is found. Skin tissue of guinea pigs in the positive control group showed substantial recovery of the skin lesion state, and showed only reddish epidermis, normal hair growth, and no dandruff. The skin of the back of the guinea pigs in the model group still has the aggregation of white scales, and obvious trichophyta rubrum characteristic colonies grow through SDA flat-plate culture. After 10 days of treatment, compared with the model group, the skin damage state is obviously recovered after the experimental group is dosed, and the skin is free from dandruff and redSwelling phenomenon, cure rate is 100%. Compared with the terbinafine cream with the cure rate of 66.7%, the experimental gel cream has better cure effect and 37.5% of score improvement (figure 8), and the result shows that the pharmaceutical preparation has excellent treatment effect on a guinea pig dermatophyte infection model and has great drug development prospect.

In summary, we have found that the fluorescent whitening agent 85 has excellent antibacterial activity against dermatophytes, particularly trichophyton rubrum and microsporum gypseum, and can exhibit remarkable antibacterial activity against drug-resistant strain candida albicans. The stilbene fluorescent whitening agent is considered to be a novel and effective antifungal compound, is developed as a novel antifungal medicament, has unique action mechanism and action target points, has industry leading property, and has huge medicament development potential and value.

Claims

1. The use of a fluorescent whitening agent in the manufacture of a medicament for inhibiting fungal activity, wherein the fluorescent whitening agent is fluorescent whitening agent 85; the fungus is drug-resistant candida albicans.