Photosensitizer for treating tinea capitis and application thereof

Abstract

The invention relates to the technical field of antifungal treatment, in particular to a photosensitizer for treating tinea capitis and application thereof.According to the photosensitizer for treating tinea capitis, microsporum canis which is the most common pathogen of tinea capitis in China is used as an observation object, and the killing effect of methylene blue (MB) with different concentrations and photodynamic force (PDT) mediated by different illumination energy densities on the microsporum canis is explored through in-vitro experiments. A microsporum canis mouse skin and hair infection model is constructed for in-vivo experiments, the curative effect is observed, related mechanisms are deeply discussed, and an experimental basis is provided for clinical methylene blue photodynamic antifungal treatment; besides, a traditional Chinese medicine photosensitizer capable of inhibiting growth of microsporum canis is screened from traditional Chinese medicines with bacteriostasis reports, and is combined with photodynamic antibacterial treatment, so that the traditional Chinese medicine composition is a safe and green therapy for effectively treating skin tinea.

Description

Technical Field

[0001] The present invention relates to the technical field of antifungal treatment, and particularly relates to a photosensitizer for treating tinea capitis and its application.

Background Art

[0002] There are more than 1.7 billion cases of dermatophytosis globally, and tinea capitis is a disease caused by dermatophyte infection of the scalp and hair. Microsporum canis is an animalophilic fungus that can infect humans, causing infectious diseases such as the scalp, hair, skin, and nails, and can also cause deep scalp infections leading to kerion. Since tinea capitis is prone to occur in children, especially preschool children, it can break out and spread in places such as kindergartens and primary schools. It is highly contagious and can even break out and cause widespread superficial mycosis in specific populations under certain conditions, seriously endangering the health of children. In recent years, the number of child patients has shown an obvious upward trend, and even small-scale outbreak events have occurred. Due to low clinical attention, atypical symptoms, and frequent misdiagnosis and missed diagnosis of tinea capitis, the course of the disease in patients is prolonged, and serious complications such as scars and permanent hair loss occur. The dermatophytes causing tinea capitis are more commonly Microsporum and Trichophyton.

[0003] The current conventional treatment plan for tinea capitis is still the five-word principle of "taking, applying, shaving, washing, and disinfecting". Among them, oral antifungal drugs are crucial. The commonly used oral drugs for tinea capitis currently are griseofulvin, terbinafine, itraconazole, etc. However, all current oral antifungal drugs have the drawback of a long treatment cycle, and long-term use may cause side effects such as abnormal liver function, headache, digestive tract symptoms, and neutropenia. The limitations of systematic use of antifungal drugs prompt us to seek safer and more effective new treatment methods.

[0004] Antimicrobial photodynamic therapy (aPDT) is an effective alternative or combined treatment strategy. aPDT uses photosensitizers (PS), which produce reactive oxygen species under visible light irradiation and kill microorganisms through non-specific oxidative damage. The three core elements in photodynamic therapy (PDT) are: photosensitizer, light source, and target tissue. PDT has a wide range of activities and can kill all known types of microorganisms. Compared with antibacterial and antifungal drugs, it rarely induces drug resistance, does not damage the surrounding host tissues, and the treatment is targeted. It is one of the very promising new therapies in anti-infection treatment.

[0005] There are a wide variety of photosensitizers, including aminolevulinic acid, porphyrin, phenothiazine, etc. Methylene Blue (MB) is the most studied drug among thiazine photosensitizers and has been widely used in clinical work such as detoxification, analgesia, and staining markers. Due to the advantages of MB such as economy, no need for light avoidance, wide absorption spectrum, and safety, it has become one of the hotspots in photodynamic therapy research. The widespread clinical use has confirmed that MB has the characteristics of low toxicity and high safety. MB has good water solubility, and its maximum absorption peak in water is at the red light wavelength of 668nm. Red light has a penetration depth twice that of blue light and can penetrate 3mm of human tissue, so it has more treatment advantages. Therefore, when using PDT mediated by phenothiazine photosensitizers to treat fungal infections in human body cavity parts, skin, nails and other organs, it has a better ability to clear local fungi. At the same time, MB also has the characteristics of low price, oxidation-reduction property, high safety, and relatively stable properties in the air.

[0006] Traditional Chinese medicine photosensitizers have also been studied in the prior art: Traditional Chinese medicine photosensitizers have the characteristics of high production of singlet oxygen free radicals, high phototoxicity, low dark toxicity, and good tissue penetration. They are relatively ideal photosensitizers and an important part of photodynamic therapy. However, there are a wide variety of traditional Chinese medicine photosensitizers, with different advantages and disadvantages, making it difficult to choose clinically.

[0007] Therefore, in order to find various photosensitizers suitable for inhibiting Microsporum canis infection, and combining photodynamic antimicrobial therapy is a safe and green therapy for effectively treating tinea corporis.

Summary of the Invention

[0008] In view of the above, it is necessary to find various photosensitizers suitable for inhibiting Microsporum canis infection, and combining photodynamic antimicrobial therapy is a safe and green therapy for effectively treating tinea corporis.

[0009] To achieve the above object, the technical solution adopted by the present invention is:

[0010] A photosensitizer for treating tinea capitis, wherein the photosensitizer is methylene blue.

[0011] Further, the photosensitizer further includes Artemisia argyi extract and / or Eclipta prostrata extract.

[0012] Further, the preparation method of the Artemisia argyi extract or Eclipta prostrata extract is: Mix dry Artemisia argyi or dry Eclipta prostrata with a solid-liquid mass ratio of 1:1 and boil. After boiling, keep boiling for 30 minutes, then filter to obtain the filtrate and concentrate it. Concentrate the filtrate to 1 / 10 of the original solution to obtain the corresponding extract.

[0013] The present invention also includes the application of the photosensitizer in the preparation of an inhibitor for inhibiting Microsporum canis.

[0014] The present invention also includes the application of the photosensitizer in the preparation of drugs for treating tinea capitis.

[0015] Furthermore, for different red light irradiation energy groups of methylene blue: 70W / J, 80W / J, 90W / J, 100W / J, 110W / J, 120W / J, 130W / J, 140W / J, 150W / J, the average MIC values under the irradiation conditions are respectively: 1.031ug / ml, 0.523ug / ml, 0.367ug / ml, 0.281ug / ml, 0.297ug / ml, 0.281ug / ml, 0.289ug / ml, 0.305ug / ml, 0.297ug / ml, 0.289ug / ml.

[0016] Furthermore, for different red light irradiation energy groups of Artemisia argyi extract: 70W / J, 80W / J, 90W / J, 100W / J, 110W / J, 120W / J, 130W / J, 140W / J, 150W / J, the average MIC values under the irradiation conditions are respectively: 32mL / 100mL, 32mL / 100mL, 16mL / 100mL, 16mL / 100mL, 16mL / 100mL, 8mL / 100mL, 8mL / 100mL, 32mL / 100mL, 32mL / 100mL; for different red light irradiation energy groups of Eclipta prostrata extract: 60W / J, 70W / J, 80W / J, 90W / J, 100W / J, 110W / J, 120W / J, 130W / J, 140W / J, 150W / J, the average MIC values under the irradiation conditions are respectively 32mL / 100mL, 32mL / 100mL, 16mL / 100mL, 16mL / 100mL, 16mL / 100mL, 8mL / 100mL, 8mL / 100mL, 4mL / 100mL, 16mL / 100mL, 16mL / 100mL.

[0017] The present invention has the following beneficial effects:

[0018] This application takes Microsporum canis, the most common pathogenic bacterium of tinea capitis in China, as the observation object, and explores the killing effect of different concentrations of methylene blue (Methylene Blue, MB) and photodynamic therapy (Photodynamictherapy, PDT) mediated by different light energy densities on Microsporum canis through in vitro experiments. A mouse skin and hair infection model of Microsporum canis is constructed for in vivo experiments to observe the curative effect and deeply explore its related mechanism, providing an experimental basis for clinical methylene blue photodynamic antifungal treatment; in addition, traditional Chinese medicine photosensitizers that can inhibit the growth of Microsporum canis are screened from traditional Chinese medicines with antibacterial reports, and combined with photodynamic antibacterial treatment, it is a safe and green therapy for effectively treating tinea corporis.

Description of the Drawings

[0019] Figure 1 It is a schematic diagram of animal experiments.

[0020] Figure 2 It is

[0021] Figure 3 It is a statistical box plot of methylene blue on human dermal fibroblasts (HDF-a).

[0022] Figure 4 It is a bar chart of the survival rate and mortality rate of methylene blue on human dermal fibroblasts (HDF-a) at each concentration of MB; the y-axis of the bar chart represents the survival or mortality rate. The x-axis represents different treatments. Red represents the survival rate and blue represents the mortality rate.

[0023] Figure 5 It is a statistical box plot of methylene blue on human immortalized epidermal cells (HACAT).

[0024] Figure 6 It is a bar chart of the survival rate and mortality rate of methylene blue on human immortalized epidermal cells (HACAT) at each concentration of MB; the y-axis of the bar chart represents the survival or mortality rate. The x-axis represents different treatments. Red represents the survival rate and blue represents the mortality rate.

Detailed implementation manners

[0025] All the features disclosed in this specification, or all the steps in the disclosed methods or processes, except for mutually exclusive features and / or steps, can be combined in any way.

[0026] Any feature disclosed in this specification (including any additional claims, abstract), unless specifically stated, each feature is only an example of a series of equivalent or similar features.

[0027] Example 1:

[0028] This example studies the in vitro killing effect of methylene blue photodynamic therapy on Microsporum canis. The specific experimental protocol and results are as follows:

[0029] 1. Experimental strains: 24 strains of Microsporum canis were all isolated from the clinical isolates of Microsporum canis from tinea capitis patients who visited the dermatology and venereology outpatient department of the First Affiliated Hospital of Guangxi Medical University from January 1, 2019 to May 31, 2022. All were confirmed as Microsporum canis through morphological and molecular identification.

[0030] 2. Preparation of Microsporum canis suspension: All the test strains were inoculated on PDA medium and cultured at 28 °C for 10 - 14 d to activate them. The suspensions of all spores and short hyphal fragments were sucked into a sterile test tube and mixed evenly. The concentration of the suspension was adjusted to 2×10 5 CFU / mL - 3×10 5CFU / mL. Previous studies have shown that fungal hyphae segments also have growth and invasive properties. Therefore, this study used a mixture of Microsporum canis and hyphae as the research object, and tried to oscillate the hyphae into short hyphae. The spore suspension used in the drug sensitivity test should be prepared and used immediately.

[0031] 3. Inoculation and cultivation of bacterial suspension: Use RPMI-1640 liquid culture medium to dilute the prepared spore suspension 50 times to obtain an inoculated bacterial suspension equivalent to 2 times the final concentration, so that the concentration of Microsporum canis is 1×10 3 -3×10 3 CFU / mL. Then, vortex the bacterial solution to mix it evenly, and add 100 μL of the bacterial suspension of the corresponding strain to the 1st to 10th columns of wells (gradient drug wells) and the 11th column of wells (positive wells) of the drug sensitivity plate, so that the final concentrations of Microsporum canis and the quality control strain in the drug sensitivity plate are 1×10 3 -3×10 3 CFU / mL, 0.5×10 3 -2.5×10 3 CFU / mL. To avoid cross contamination, each drug sensitivity plate was inoculated with only the same fungal suspension. After the bacterial suspension was inoculated, the drug sensitivity plate was placed in a 28°C incubator without shaking and the drug sensitivity results were observed after 72 hours.

[0032] 4. In vitro antibacterial test and determination of minimum inhibitory concentration: 1% MB was diluted with sterile water to 48ug / mL, 24ug / mL, 12ug / mL, 6ug / mL, 3ug / mL, 1.5ug / mL, 0.75ug / mL, 0.375ug / mL, 0.1875ug / mL, 0.09375ug / mL, negative control wells, and positive control wells. Add the prepared Microsporum canis, and incubate the photosensitizer and fungus in the dark for 20 minutes; the negative control group was diluted with 1640 medium to 1×10 4 -7×10 4 CFU / mL working solution; each of the above groups was inoculated into a 96-well plate with 100ul of working solution per well using a micropipette gun, with 3 replicate wells for each group; the MB-PDT group was given red light (wavelength 632±10nm, energy density set at 60J-150J / cm 2 ) irradiation, MB group was added with different concentrations of MB solution and then wrapped with tin foil to avoid light, and the negative control group was also protected from light; after the MB-PDT group was irradiated, the 96-well plate was placed in a dark aerobic environment in a 26°C incubator and cultured. The negative control group was observed for 72 hours and the good growth was considered the MIC value. No visible growth under a microscope was considered the MIC value. This experiment was repeated 3 times.

[0033] 5. Killing effects of MB-PDT mediated by different light energies on Microsporum canis: According to different light energy densities, it was divided into 60 J / cm 2 , 70 J / cm 2 , 80 J / cm 2 , 90 J / cm 2 , 100 J / cm 2 , 100 J / cm 2 , 110 J / cm2, 120 J / cm 2 , 130 J / cm 2 , 140 J / cm 2 , 150 J / cm 2 And a blank control group and a single light irradiation group were set up. The effects of MB-PDT with different energy densities on Microsporum canis were measured, and the experiment was repeated three times.

[0034] Reference to the CLSIM38-A2 protocol: Determine the minimum inhibitory concentration (MIC) value of methylene blue photodynamic killing of Microsporum canis; culture Microsporum canis in a 96-well plate and irradiate it with red light with a light source wavelength of 632 ± 10 nm to detect the effects of different MB concentrations and different light energy densities on the biological activity of Microsporum canis. In vitro drug sensitivity shows that there are relatively few current studies on MB-PDT against Microsporum canis, and only a small number of case reports can be seen. There is currently no unified standard for the effective concentration of MB, the light source, and the light energy density. The experimental results of each experimental group in this experiment show that MB-PDT has a good effect on killing the biological activity of Microsporum canis in vitro. During the experiment, we initially referred to the conventional drug concentration for in vitro experiments (the MB concentration was set at 96 μg / ml - 1.5 μg / ml), and it was found that all Microsporum canis irradiated with energy were inhibited. Therefore, the MB drug concentration was gradually diluted down to 48 μg / ml - 0.09375 μg / ml, which had a good killing effect on Microsporum canis. No obvious killing effect on Microsporum canis was seen in the single MB group, but the growth was relatively slow compared to the negative control group. As an oxidation-reduction type photosensitizer, MB may affect the liquid medium and thus affect the growth of fungi. The negative control group (only the red light irradiation group) had no obvious killing effect on Microsporum canis. The growth of the negative control group in this experiment was good. The MIC means of different irradiation energy groups (60 W / J, 70 W / J, 80 W / J, 90 W / J, 100 W / J, 110 W / J, 120 W / J, 130 W / J, 140 W / J, 150 W / J) in the MB-PDT group were (1.031 μg / ml, 0.523 μg / ml, 0.367 μg / ml, 0.281 μg / ml, 0.297 μg / ml, 0.281 μg / ml, 0.289 μg / ml, 0.305 μg / ml, 0.297 μg / ml, 0.289 μg / ml). Conclusion: MB-PDT can effectively kill Microsporum canis in vitro; within a certain range, the killing effect of MB-PDT is positively correlated with the MB concentration and the light energy density.

[0035] Example 2:

[0036] This example studies the effect of methylene blue photodynamic on the ultrastructure of Microsporum canis, as follows:

[0037] 1. In vitro experiment of methylene blue photodynamic on Microsporum canis: The in vitro experiment of methylene blue photodynamic on Microsporum canis is the same as that in Example 1.

[0038] 2. Transmission electron microscopy specimen preparation steps:

[0039] (1) Sampling and fixation: Centrifuge to collect cell or bacterial precipitate, and the precipitate should be at least the size of a mung bean. Remove the culture medium, add electron microscopy fixative, resuspend and mix well at 4°C for 2 - 4 h, and fix, store and transport at 4°C.

[0040] (2) Agar pre-embedding: Centrifuge the cells or bacteria, discard the supernatant, add 0.1 M phosphate buffer PB (pH 7.4), mix well and rinse for 3 min, then centrifuge again. Repeat the washing 3 times. Prepare a 1% agarose solution by heating and dissolving in advance. After cooling slightly, add it into an EP tube. Before the agarose solidifies, pick up the precipitate with forceps and suspend it in the agarose.

[0041] (3) Post-fixation: Fix with 1% osmium tetroxide prepared with 0.1 M phosphate buffer PB (pH 7.4) in the dark at room temperature for 2 h. Rinse 3 times with 0.1 M phosphate buffer PB (pH 7.4), 15 min each time.

[0042] (4) Dehydration at room temperature: The tissue is successively dehydrated in 30% - 50% - 70% - 80% - 95% - 100% - 100% alcohol for 20 min each time, and in 100% acetone twice, 15 min each time.

[0043] (5) Infiltration and embedding: Acetone : 812 embedding agent = 1 : 1 at 37°C for 2 - 4 h, acetone : 812 embedding = 1 : 2 at 37°C for overnight infiltration, and pure 812 embedding agent at 37°C for 5 - 8 h. Pour the pure 812 embedding agent into the embedding plate, insert the sample into the embedding plate, and place it in an oven at 37°C overnight.

[0044] (6) Polymerization: Place the embedding plate in an oven at 60°C for polymerization for 48 h, and take out the resin block for standby.

[0045] (7) Ultra-thin sectioning: Cut ultra-thin sections of 60 - 80 nm from the resin block with an ultra-microtome, and pick up the sections with a 150-mesh Fanghua membrane copper grid.

[0046] (8) Staining: Stain the copper grid with 2% saturated alcoholic solution of uranyl acetate in the dark for 8 min; wash 3 times with 70% alcohol; wash 3 times with ultrapure water; stain with 2.6% lead citrate solution in the absence of carbon dioxide for 8 min; wash 3 times with ultrapure water and gently blot with filter paper. Place the copper grid sections in a copper grid box and dry at room temperature overnight. Observe under a transmission electron microscope and collect and analyze the images.

[0047] The experimental results are as follows: When the drug concentration is 0.1875 ug / mL, the degree of bacterial damage is relatively mild, the electron density of intracellular protoplasm decreases and becomes uniform, and the structure of cell organelles is relatively clear and acceptable. Under the action of the drug at a concentration of 0.375 ug / mL, the degree of bacterial damage is relatively slight, with local dissolution or lightening of intracellular protoplasm, and most of the mitochondria have blurred structures and reduced cristae. Under the action of the drug at a concentration of 0.75 ug / mL, the degree of bacterial damage is relatively slight, with local dissolution or lightening of intracellular protoplasm, and most of the mitochondria have blurred structures and reduced cristae. At the MB drug concentration of 1.5 ug / ml, the degree of bacterial damage is relatively severe, with large-area dissolution of intracellular protoplasm, disintegration of mitochondria, uneven cell wall thickness, and local blurring. In the experiment, most Microsporum canis were killed after MB-PDT treatment at a concentration greater than 1.5 ug / ml. It was difficult to collect enough bacteria for transmission electron microscopy detection during the experiment. Therefore, in this experiment, only Microsporum canis after PDT with low drug concentrations of 1.5 ug / ml, 0.75 ug / ml, 0.375 ug / ml, and 0.1875 ug / ml MB could be observed by transmission electron microscopy. The results showed that under the action of MB-PDT at a concentration of 1.5 ug / ml, mitochondria disintegrated, the membrane was damaged in a large area, the matrix was dissolved and overflowed in a large area, and a large number of cristae were broken and dissolved; individual lipid droplets were visible and irregular, without fusion, the cell wall had uneven thickness, and local blurring. The damage decreased in turn at the MB drug concentrations of 0.75 ug / ml, 0.375 ug / ml, and 0.1875 ug / ml, indicating that within a certain range, the greater the drug concentration, the more severe the damage to Microsporum canis. The main site of damage was the mitochondria, followed by the cell wall. This provided a preliminary basis for subsequent exploration of the mechanism of MB-PDT killing Microsporum canis.

[0048] Example 3:

[0049] This example studied the effects of methylene blue on the viability of human skin fibroblasts and human immortalized epidermal cells, specifically as follows:

[0050] 1. CCK-8 cytotoxicity detection:

[0051] (1) Inoculate the cells into a 96-well plate, with 100 μl of the counted and diluted cell suspension in each well, so that the number of cells in each well is 3000 cells / well.

[0052] (2) Place the seeded plate of cells into the incubator and continue to culture for 24 hours to allow the cells to adhere completely.

[0053] (3) Set up a blank group (only the culture medium), a control group (culture medium + cells), and an experimental group (culture medium + cells + drug) respectively. The drug concentrations are 48 μg / mL, 24 μg / mL, 12 μg / mL, 6 μg / mL, 3 μg / mL, 1.5 μg / mL, 0.75 μg / mL, 0.375 μg / mL, 0.1875 μg / mL, and 0.09375 μg / mL respectively. Set up 3 replicate wells for each group. Use a pipette to aspirate the original culture medium completely, replace it with different culture media according to the grouping, and put it into the incubator for continued culture for 2.5 hours.

[0054] (4) Use a pipette to completely aspirate the liquid in the well plate, replace it with DMEM medium without fetal bovine serum, and carefully add 10 μL of CCK8 solution to each well (pay attention not to generate bubbles in the well). After adding the CCK8 solution, continue to put the culture plate into the incubator for incubation for 2.5 hours.

[0055] (5) After incubation, use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance at 450 nm in each well.

[0056] (5) Calculate the OD value and average value of each well, and calculate the cells. The formula is as follows:

[0057] Cell viability (%) = [A (experimental) - A (blank group)] / [A (negative control) - A (blank)] × 100%.

[0058] Inhibition rate (%) = [A (negative control) - A (experimental)] / [A (negative control - A (blank)] × 100%

[0059] 2. Calculate IC 50 、IC 10 And use Graphpad prism to plot the dose-response curve:

[0060] (1) Use Graphpad prism software, select "XY" for the chart type, check "Enter or import data into a new table" in the Data table option; check "Enter 3 replicate values in side-by-side subcolumns" in the Options option. In this example, there are 3 replicate wells for the drug concentration, so enter the number "3". After setting, click "Creat" to create the chart.

[0061] (2) Transform the data and analyze the data to finally obtain the dose-response curves.

[0062] The experimental results are as follows:

[0063] 3. Effects of methylene blue on the survival rate and inhibition rate of human skin fibroblasts and human immortalized epidermal cells:

[0064] To prove the effects of methylene blue at different concentrations on the viability of human skin fibroblasts (HDF-a) and human immortalized epidermal cells (HACAT), we used CCK-8 to detect the survival rate and proliferation inhibition rate of human skin fibroblasts and human immortalized epidermal cells. Different concentrations of MB (48 μg / ml, 24 μg / ml, 12 μg / ml, 6 μg / ml, 3 μg / ml, 1.5 μg / ml, 0.75 μg / ml, 0.375 μg / ml, 0.1875 μg / ml, 0.09375 μg / ml) were set to observe their effects on the viability of human skin fibroblasts and human immortalized epidermal cells at different action durations. Moreover, as the concentration of MB decreased, the survival rate of human skin fibroblasts and human immortalized epidermal cells gradually increased, and the inhibition rate gradually decreased.

[0065] Table 1 Inhibition rate and survival rate of human skin fibroblasts treated with methylene blue at different concentrations

[0066]

[0067] (2) Effects of methylene blue on human immortalized epidermal cells (HACAT) are shown in Table 2 as follows:

[0068] Table 2 Survival rate and inhibition rate of human immortalized epidermal cells treated with methylene blue at different concentrations

[0069]

[0070]

[0071] (3) Effects of methylene blue on human skin fibroblasts (HDF-a) are as follows: Figures 3 - 4 shown:

[0072] Figure 3This is a statistical box plot of methylene blue on human dermal fibroblasts (HDF-a). In the figure: the letter c represents the control group, bk represents the blank group, and the MB concentrations corresponding to the 1-10 distribution are 48 μg / ml, 24 μg / ml, 12 μg / ml, 6 μg / ml, 3 μg / ml, 1.5 μg / ml, 0.75 μg / ml, 0.375 μg / ml, 0.1875 μg / ml, 0.09375 μg / ml). The solid line in the box plot represents the median of each group of data. The upper and lower ends of the box plot represent the 25% and 75% percentiles, and the whiskers outside the box represent the upper and lower limits of the data respectively. One-way ANOVA and multiple comparisons were used to analyze the data of different treatments. Different lowercase letters indicate significant differences at the 0.05 level in the experimental interval of the same variable (p < 0.05). It can be seen from the figure that there is no significant statistical difference in the MB concentration of 48 μg / ml - 6 μg / ml compared with the blank group, while there is a significant statistical difference in the MB concentration of 3 μg / ml - 0.09375 μg / ml. Figure 4 This is a bar chart of the survival rate and mortality rate of methylene blue on human immortalized epidermal cells (HACAT) at each concentration: In the figure, the y-axis of the bar chart represents the survival or mortality rate. The x-axis represents different treatments. Red represents the survival rate, and blue represents the mortality rate.

[0073] (4) The effect of methylene blue on human immortalized epidermal cells (HACAT) is specifically as Figures 5 - 6 shown:

[0074] Figure 5 This is a statistical box plot of methylene blue on human immortalized epidermal cells (HACAT). In the figure, the letter c represents the control group, bk represents the blank group, and the MB concentrations corresponding to the 1-10 distribution are 48 μg / ml, 24 μg / ml, 12 μg / ml, 6 μg / ml, 3 μg / ml, 1.5 μg / ml, 0.75 μg / ml, 0.375 μg / ml, 0.1875 μg / ml, 0.09375 μg / ml). The solid line in the box plot represents the median of each group of data. The upper and lower ends of the box plot represent the 25% and 75% percentiles, and the whiskers outside the box represent the upper and lower limits of the data respectively. One-way ANOVA and multiple comparisons were used to analyze the data of different treatments. Different lowercase letters indicate significant differences at the 0.05 level in the experimental interval of the same variable (p < 0.05). It can be seen from the figure that there are significant statistical differences in the MB concentration of 48 μg / ml - 0.09375 μg / ml compared with the blank group and the control group. Figure 6 This is a bar chart of the survival rate and mortality rate of methylene blue on human immortalized epidermal cells (HACAT) at each concentration. In the figure, the y-axis of the bar chart represents the survival or mortality rate. The x-axis represents different treatments. Red represents the survival rate, and blue represents the mortality rate.

[0075] In summary, the research of this application found that the IC 10 of MB on human dermal fibroblasts (HDF-a) is 0.4487 μg / ml; the IC 10 of MB on human immortalized epidermal cells (HACAT) is 0.7738 μg / ml. Therefore, it can be considered that when the MB concentration ≤ 0.4487 μg / ml, there is no obvious toxicity to human dermal fibroblasts (HDF-a); when the MB concentration ≤ 0.7738 μg / ml, there is no obvious toxicity to human immortalized epidermal cells (HACAT) and the safety is good.

[0076] Example 4:

[0077] This example is a study on the efficacy of methylene blue photodynamic therapy on a murine model of Microsporum canis infection, which is as follows:

[0078] In this experiment, a murine skin and hair infection model was innovatively established, and methylene blue photodynamic therapy was carried out using this model, providing a theoretical basis for the application of antibacterial photodynamic therapy in clinical practice. (This animal experiment protocol has passed the animal experiment ethical review of Guangxi Medical University, ethical number: 202303011).

[0079] 1. Establishment of a murine skin and hair (Microsporum canis) infection model:

[0080] (1) Experimental animals: Ordinary healthy male Kunming mice, 8 weeks old, weighing 35 - 40 g, provided by the Animal Experiment Center of Guangxi Medical University.

[0081] (2) Microsporum canis: Provided by the Dermatology Mycology Laboratory of the First Affiliated Hospital of Guangxi Medical University.

[0082] Preparation of Microsporum canis suspension: All test strains were inoculated on PDA medium and cultured at 28 °C for 10 - 14 d to activate them. The suspensions of all spores and hyphal fragments were aspirated into a sterile test tube and mixed evenly. The spore suspensions of each strain were counted using a cell counting chamber, and the spore suspension concentration of each strain was adjusted to 1×10 9 CFU / mL with sterile water and reserved for use. Observe the colony development and microscopic morphology, and subculture on the medium for 2 times to ensure its viability.

[0083] (3) Method for constructing animal model: The mice were fed freely in the experimental environment for one week to adapt to the experimental environment; the mice were anesthetized by inhaling isoflurane. After about 5 minutes, the body of the mice became soft and there was a blinking reaction. Pay attention to keeping the mice warm during the experiment. Use an electric razor to shave the hair on the back of the mice, try to take a large area, and the shaving area is about 5 cm × 3 cm in size. Apply depilatory cream evenly on the shaved area. After 5 - 8 minutes, gently scrape off the surface hair without scratching the surface skin. Dip a sterile cotton swab in physiological saline to clean the hair on the back of the mice, and disinfect the back with 75% alcohol. After the skin at the disinfected area is dry, use a sterile rolling needle to scrape and polish the back area until punctate bleeding is appropriate; disinfect again with 75% alcohol; after the disinfected area is dry, take the prepared bacterial suspension and apply it evenly on the damaged skin surface. In order to improve the success rate of model establishment, use the bacterial suspension to wet compress the back of the mice, and use an electric blanket to keep the mice warm. Inoculate again the next day using the same method; raise the mice inoculated with Microsporum canis in a standard environment. Observe the mental state, appetite, and weight of the mice every day after inoculation, observe the manifestations of the skin at the inoculation site, and make records every day. Starting from 7 days after inoculation, perform fungal fluorescence and culture on the scabs, erythema, and papules that appear at the inoculation site every day. The culture method is as follows: A. Place the scales or broken hairs on a glass slide, add a drop of 10 - 20% potassium hydroxide solution, cover with a coverslip, and observe under a microscope after a few minutes; then add a drop of fungal fluorescence staining solution, cover with a coverslip, and observe under a fluorescence microscope after a few minutes. The fungal components (spores or hyphae) show blue or green fluorescence, which is clearer. B. Inoculate the broken hairs or scalp scales on PDA and culture at 25℃ - 28℃ for 2 - 4 weeks. Observe once a day. If there is no colony growth after 4 weeks, it is negative. Identify the fungal species according to the colony morphology and microscopic structure of the cultured fungi.

[0084] (4) Pathological tissue and PAS staining evaluation of the Microsporum canis-infected mouse model: On the 14th day after inoculating Microsporum canis into mice, one mouse from the experimental group and one mouse from the control group were randomly selected, anesthetized and sedated with an overdose of isoflurane, and then sacrificed. The skin was disinfected with 75% alcohol. A fusiform incision about 1 cm × 1.5 cm was made at the lesion site with a sterile scalpel. After the skin was picked up with forceps and separated from the subcutaneous tissue, it was placed in formaldehyde for fixation and preservation. The fixed tissue block was cut into 1 cm × 1 cm with a surgical blade. The tissue block after washing with water was dehydrated with alcohol of different concentrations, infiltrated with melted paraffin, and then embedded. The embedded tissue block was cut open with a knife, the remaining wax was trimmed, and the four sides and the bottom were cut flat. After heating the wax block to make it adhere closely to the tissue frame, it was finally fixed on the microtome for sectioning. The cut sections were placed in water to flatten them, picked up with a glass slide, and then flattened in warm water. The dried paraffin sections were sequentially passed through xylene I and xylene II (each for 10 min), different gradient alcohols (100% for 1 min, twice; 95% for 1 min, twice; 85% for 1 min; 80% for 1 min), and finally placed in distilled water for 1 min for dewaxing. The dewaxed tissue sections were stained with hematoxylin solution for 10 min, washed with running water for 1 min to remove the hematoxylin solution, soaked in 1% hydrochloric acid-ethanol solution for 3 s, slightly washed with water for 2 s, blued with warm water for 5 s, washed with running water for 1 min, washed with distilled water for 1 min, then stained with 1% eosin solution for 3 min, washed with water for 2 s, dehydrated with gradient alcohol, and cleared with xylene. Sealed with neutral gum, and gently pressed with forceps to expel air bubbles. Observe the changes in the thickness and integrity of each layer of the skin under the microscope. PAS staining: The sections were dewaxed to water, rinsed with distilled water for 1 min, added acetic acid differentiation solution for 2 min, and poured off; added Alcian blue (pH 2.5) and stained for 10 min, poured off, added acetic acid differentiation solution for 2 min and poured off, rinsed with running water for 5 min; oxidized with periodic acid for 15 min, rinsed with distilled water and waited to dry; then placed in Schiff's reagent and stained at room temperature for 10 - 20 min; after rinsing with sulfurous acid solution twice, then rinsed with tap water for 10 min and waited to dry; dehydrated, cleared, and sealed. Observe the number of hyphae and spores in the skin tissue under an optical microscope to compare the infection effects.

[0085] (5) Establishment of the Microsporum canis skin and hair infection model:

[0086] ① Rash manifestations: Currently, there is no criterion for judging animal models of fungal hair infections. In this experiment, the criterion for judging animal models of skin fungal infections was referred to. When the following conditions were met, the production of the dermatophyte infection animal model was considered successful; erythema, erosion, desquamation, and crusting appeared at the inoculation site; spores or hyphae were visible by direct microscopy of samples taken from the skin lesion site; Microsporum canis was cultured from the fungus. All of the above conditions must be met for the establishment of a successful Microsporum canis skin infection model. In addition to the manifestations related to the skin infection model, the hair infection model should also have the discovery of hyphae within the hair by fungal fluorescence; dermoscopy and Wood's lamp examination; pathological examination indicators (especially the manifestation of hyphae within the hair), etc. The basis for judging the success of the fungal skin and hair infection mouse models in this experiment included: rash manifestations at the inoculation site; the presence of hyphae and spores within the hair visible by fungal fluorescence of the hair at the rash site; Microsporum canis cultured from the rash site; pathological manifestations.

[0087] (6) Fungal fluorescence and fungal culture: Hyphae and spores within the hair were visible by fungal fluorescence of the diseased hair of the mice. Microsporum canis was cultured from the scabs, scales, and other rashes at the diseased hair and rash sites of the mice.

[0088] (7) Pathological examination and PAS staining of the rash sites of the mice: Pathological examination of the rash sites of the mice showed a large number of neutrophil infiltrations around the dermis, hair follicles, and within the hair follicles, and the PAS staining was positive, with hyphae and spores within the hair visible. Pathological examinations of the livers, kidneys, and spleens of the mice showed no special abnormalities, and the PAS staining was negative.

[0089] (8) Dermoscopy of the Microsporum canis skin and hair infection model: The difference in hair diameter was less than 20%, short vellus hairs and white scales were visible, comma-like hairs, spiral-like hairs, and Morse code-like hairs were not seen.

[0090] 2. Study on the efficacy of methylene blue photodynamic therapy on the mouse skin and hair (Microsporum canis) infection model:

[0091] (1) Preparation of the photosensitizer: The prepared 1% concentration of methylene blue was added to 10 ml of moisturizing gel and diluted 10 times, and it was stored in the dark for later use.

[0092] (2) Grouping of experimental animals: The mice were divided into ① model group (untreated group after infection); ② methylene blue photodynamic therapy group; ③ terbinafine topical treatment control group (control group 1); ④ single red light irradiation treatment group (control group 2).

[0093] (3) Treatment of Microsporum canis mouse animal model with methylene blue photodynamic therapy: Mice were anesthetized by isoflurane inhalation. The prepared methylene blue gel photosensitizer was applied to the skin lesions on the back of the mice, generally spreading 1 cm beyond the lesions, covered with plastic wrap, and protected from light with black light-shielding paper for 2 hours. After the time of photosensitizer wrapping, the drugs on the back were wiped clean with normal saline, and then irradiated with red light (wavelength 632±10 nm) at an energy of 100 W / J for 20 min. Once every 3 days, for a total of 2 treatments. The specific operation is as Figures 1 - 2 shown.

[0094] (4) Terbinafine topical treatment control group (control group 1): Mice in the terbinafine topical treatment control group were topically treated with terbinafine cream every day, evenly applied to the skin and hair infection model lesions of Microsporum canis, and slightly massaged after application until the drug was absorbed, for a total of 7 days.

[0095] (5) Single red light irradiation treatment control group (control group 2): Mice in the single red light irradiation treatment control group were irradiated with a red light therapeutic instrument at an energy of 100 W / J once every 3 days, 20 min each time, for a total of 2 treatments.

[0096] (6) Efficacy observation: The changes in the skin at the inoculation site were observed daily and evaluated on the 11th and 14th days after inoculation. The evaluation contents included skin lesions and mycological examinations. Skin lesions included erythema area, severity of papules, crusts, scales, and alopecia. The judgment criteria were as follows: erythema area less than <1 / 3 of the inoculation area was 1 point, erythema area 1 / 3 - 2 / 3 was 2 points, erythema area >2 / 3 was 3 points; no obvious scales was 0 point, scale area less than <1 / 3 of the inoculation area was 1 point, scale area 1 / 3 - 2 / 3 was 2 points, scale area >2 / 3 was 3 points; no obvious crusts was 0 point, a small amount of crusts was 1 point, a medium amount of crusts was 2 points; a large amount of crusts was 3 points. Alopecia area less than <1 / 3 of the inoculation area was 1 point, alopecia area 1 / 3 - 2 / 3 was 2 points, alopecia area >2 / 3 was 3 points. The scores of various skin lesions of each group of animals were added up, expressed as X±S, and compared between groups. For each mouse, hair was scraped three times for mycological microscopy + culture for mycological examination and culture. When both direct microscopy and culture were negative, it was mycological cure. If either direct mycological microscopy or culture was positive, it was mycological non-cure. The mycological cure rates of each group were compared. The cure rates were evaluated again on the 11th and 14th days.

[0097] (7) Evaluation of mycological cure rate: The lesion site was divided into 4 quadrants of up, down, left, and right. In each quadrant, 2 - 3 hairs, scales, and crusts were taken for direct mycological microscopy and mycological culture to judge mycological cure. If direct mycological microscopy and / or culture was positive, it was mycological non-cure.

[0098] (8) Pathological observation: The mice in the methylene blue photodynamic therapy group, terbinafine treatment group, and single red light irradiation group were sacrificed on the 14th day after intervention, and pathological examinations were performed on the skin lesions to evaluate the pathological changes before and after treatment.

[0099] Experimental results:

[0100] (1) Skin lesion score: Starting from the 7th day after inoculation with Microsporum canis, erythema, scales, and crusts appeared at the fungal inoculation sites of all mice, and there was no significant difference in the severity of skin lesions. After the treatment began, the erythema, papules, scales, and crusts on the backs of the photodynamic group and the terbinafine topical treatment control group (control group 1) improved compared with before. In this study, a methylene blue photodynamic therapy efficacy evaluation was conducted using a Microsporum canis skin and hair infection model, and the topical terbinafine group was selected as control group 1, and the single red light treatment group was selected as control group 2. The clinical efficacy of the methylene blue photodynamic group was far superior to that of each control group. The efficacy of the topical terbinafine group in control group 1 was superior to that of the single red light treatment group. There was no significant change in the rash condition of the untreated group within 2 weeks.

[0101] (2) Mycological cure rate: When taking samples for fungal examination, the lesion sites were divided into 4 quadrants, and several hairs and a little scale were taken from the upper, lower, left, and right quadrants respectively for direct microscopic examination of fungi and fungal culture. If the direct microscopic examination of fungi or the fungal culture was negative, it was considered mycological cure; if the direct microscopic examination of fungi and / or the fungal culture was positive, it was considered mycological non-cure. Before treatment (the 7th day), the cure rates of the MB-PDT group (group a), control group 1 (group b), control group 2 (group c), and untreated group (group d) were all 0; on the 11th day, the cure rates of groups a, b, c, and d were 60%, 20%, 10%, and 0% respectively; on the 14th day, the cure rates of groups a, b, c, and d were 80%, 20%, 10%, and 10% respectively. Fisher's exact probability method; ▲ On the 11th day, the mycological cure rates of each group Pa.d < 0.05, Pb.d < 0.05, Pc.d < 0.05, with significant differences, ▲△Pb.C > 0.05, with no significant difference; on the 15th day, the cure rates of each group Pa.b < 0.05, Pa.C < 0.05, Pa.d < 0.05, with significant differences, Pb.C.d > 0.05, with no significant difference, as shown in Table 3 specifically.

[0102] Table 3 Cure rates after methylene blue photodynamic therapy and control group treatment for Microsporum canis skin and hair infection model

[0103]

[0104] 3. Pathological observation: After the 14-day experiment, the mice in each group were sacrificed and subjected to pathological analysis. It was found that the skin of the mice in the methylene blue photodynamic group basically returned to normal, with only a small amount of inflammatory cell infiltration. The external terbinafine control group also showed improvement, and the inflammatory cell infiltration improved compared with that before treatment. However, the pathology of the single red light irradiation group still showed a large number of neutrophil infiltrations around the hair follicles.

[0105] Example 5:

[0106] This example mainly selected the photodynamic killing effect of the extracts of Polygonum multiflorum Thunb., Artemisia argyi Levl. et Vant., and Eclipta prostrata L. on Microsporum canis. The specific experimental protocol and results are as follows:

[0107] The experimental protocol referred to Example 1:

[0108] The difference is that the in vitro antibacterial agent test method is different:

[0109] (1) Preparation of the extracts of Polygonum multiflorum Thunb., Artemisia argyi Levl. et Vant., and Eclipta prostrata L.: The dry Polygonum multiflorum Thunb., dry Artemisia argyi Levl. et Vant., or dry Eclipta prostrata L. were respectively mixed and boiled according to a solid-liquid mass ratio of 1:1. After boiling, keep boiling for 30 min, and then filter to obtain the filtrate and concentrate it. Concentrate the filtrate to 1 / 10 of the original solution to obtain the corresponding extract;

[0110] (2) Dilute the corresponding extract with sterile water to 1 mL / 100 mL, 2 mL / 100 mL, 4 mL / 100 mL, 8 mL / 100 mL, 16 mL / 100 mL, 32 mL / 100 mL, 64 mL / 100 mL, negative control wells, and positive control wells. Add the prepared Microsporum canis, and co-incubate the photosensitizer and the fungus in the dark for 20 min; the negative control group was diluted to 1×10 4 -7×10 4 CFU / mL working solution; Add the working solution of each group above to the 96-well plate with a micropipette at 100 μL per well, and set 3 replicates for each group; The traditional Chinese medicine-PDT group was irradiated with red light (wavelength 632 ± 10 nm, energy density set at 60 J - 150 J / cm 2 )), and the traditional Chinese medicine extract group was wrapped with tin foil to avoid light after adding different concentrations of extract solutions. The negative control group was also treated to avoid light; After irradiation, the 96-well plate of the traditional Chinese medicine-PDT group was placed in a 26 °C incubator in a dark aerobic environment for 72 hours until the growth of the negative control group was good. The MIC value was observed under the microscope when there was no visible growth to the naked eye. This experiment was repeated 3 times.

[0111] The results obtained are as follows:

[0112] All experimental groups of the Polygonum multiflorum Thunb. extract had no obvious killing effect on Microsporum canis;

[0113] The positive extracts of Artemisia argyi extract and Eclipta prostrata extract have no killing effect on Microsporum canis:

[0114] The mean MIC values of different irradiation energy groups (60W / J, 70W / J, 80W / J, 90W / J, 100W / J, 110W / J, 120W / J, 130W / J, 140W / J, 150W / J) of Artemisia argyi extract are (no antibacterial effect, 32mL / 100mL, 32mL / 100mL, 16mL / 100mL, 16mL / 100mL, 16mL / 100mL, 8mL / 100mL, 8mL / 100mL, 32mL / 100mL, 32mL / 100mL).

[0115] The mean MIC values of different irradiation energy groups (60W / J, 70W / J, 80W / J, 90W / J, 100W / J, 110W / J, 120W / J, 130W / J, 140W / J, 150W / J) of Eclipta prostrata extract are (32mL / 100mL, 32mL / 100mL, 16mL / 100mL, 16mL / 100mL, 16mL / 100mL, 8mL / 100mL, 8mL / 100mL, 4mL / 100mL, 16mL / 100mL, 16mL / 100mL).

[0116] Example 6:

[0117] According to the experimental results of Example 5, we know that: after being mediated by light, the positive extracts of Artemisia argyi extract and Eclipta prostrata extract have antibacterial effects on Microsporum canis. Therefore, we selected these two extracts for animal experiments to study their therapeutic effects on tinea capitis after being mediated by light, as follows:

[0118] The animal experiment refers to Example 4:

[0119] (1) Preparation of photosensitizer in this example: Add 10 ml of moisturizing gel to 8mL / 100mL of Artemisia argyi extract and keep it in the dark for later use. Add 10 ml of moisturizing gel to 8mL / 100mL of Eclipta prostrata extract and keep it in the dark for later use.

[0120] (2) Grouping of experimental animals: Divide the mice into ① model group (untreated group after infection); ② Artemisia argyi photodynamic therapy group; ③ Eclipta prostrata photodynamic therapy group; ③ terbinafine topical treatment control group (control group 1); ④ single red light irradiation treatment group (control group 2); the irradiation energy is 110W / J.

[0121] Other experimental steps and evaluation methods refer to Example 4, and the results obtained are as follows:

[0122] (1) Skin lesion score: Starting from the 7th day after inoculation with Microsporum canis, erythema, scales, and crusts appeared at the fungal inoculation sites of all mice, and there was no significant difference in the severity of skin lesions. After the treatment began, the conditions of erythema, papules, scales, and crusts on the backs of the photodynamic group and the terbinafine topical treatment control group (control group 1) improved compared with before. In this study, a Microsporum canis skin and hair infection model was used to evaluate the efficacy of methylene blue photodynamic therapy, and the topical terbinafine group was selected as control group 1, and the single red light treatment group was selected as control group 2. The photodynamic therapy groups of Eclipta prostrata and Artemisia argyi both had clinical efficacy, and far exceeded each control group. The efficacy of the topical terbinafine group in control group 1 was better than that of the single red light treatment group. There was no significant change in the rash condition of the untreated group within 2 weeks.

[0123] (2) The mycological cure rate is specifically shown in Table 4:

[0124] When taking samples for fungal examination, the lesion sites were divided into 4 quadrants, and several hairs and a little scale were taken from the upper, lower, left, and right quadrants respectively for direct microscopic examination of fungi and fungal culture. If the direct microscopic examination of fungi or the culture of fungi was negative, it was considered mycological cure, and if the direct microscopic examination of fungi and / or the culture was positive, it was considered mycological non-cure. Before the treatment (the 7th day), the cure rates of the Artemisia argyi - PDT group (group a), the Eclipta prostrata - PDT group (group b), control group 1 (group c), control group 2 (group d), and the untreated group (group e) were all 0.

[0125] Table 4 Cure rates after treatment of Microsporum canis skin and hair infection model with traditional Chinese medicine photodynamic therapy and control groups

[0126]

[0127] As can be seen from Table 4, on the 11th day, the cure rates of groups a, b, c, d, and e were 80%, 70%, 20%, 10%, and 0% respectively; on the 14th day, the cure rates of groups a, b, c, d, and e were 90%, 80%, 20%, 10%, and 10% respectively.

[0128] 3. Pathological observation: After the 14-day experiment ended, the mice in each group were sacrificed and subjected to pathological analysis. It was found that the skin of the mice in the Artemisia argyi photodynamic group and the Eclipta prostrata photodynamic group basically returned to normal, with only a little inflammatory cell infiltration. The topical terbinafine control group also improved, and the inflammatory cell infiltration improved compared with before treatment. However, the pathology of the single red light irradiation group still showed a large number of neutrophil infiltrations around the hair follicles.

[0129] In summary, the methylene blue photodynamic group, the Artemisia argyi photodynamic group, and the Eclipta prostrata photodynamic group of this application can all inhibit the growth of Microsporum canis, and can combine photodynamic therapy to treat tinea capitis caused by Microsporum canis infection, which is a safe and green treatment method for tinea capitis.

[0130] The above-described embodiments merely represent several implementation manners of the present invention. Their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all fall within the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the appended claims.

Claims

1. A photosensitizer for treating tinea capitis, characterized in that: The photosensitizer is methylene blue.

2. The photosensitizer according to claim 1, characterized in that The photosensitizer also includes wormwood leaf extract and / or Eclipta prostrata extract.

3. The photosensitizer according to claim 2, characterized in that The preparation method of the wormwood leaf extract or the Eclipta prostrata extract is: dry wormwood leaf or dry Eclipta prostrata are mixed and boiled in a solid-liquid mass ratio of 1:1, and the mixture is kept boiling for 30 minutes after boiling, and then the filtrate is filtered and concentrated, and the filtrate is concentrated to 1 / 10 of the original solution to obtain the corresponding extract.

4. Use of the photosensitizer according to claim 1 or claim 2 in the preparation of an inhibitor for inhibiting Microsporum canis.

5. Use of the photosensitizer according to claim 1 or claim 2 in the preparation of a drug for treating tinea capitis.

6. Use of the photosensitizer according to claim 1 in the preparation of an inhibitor for inhibiting Microsporum canis, characterized in that: The MIC means of the methylene blue under different red light irradiation energy groups: 70W / J, 80W / J, 90W / J, 100W / J, 110W / J, 120W / J, 130W / J, 140W / J, and 150W / J irradiation conditions were: 1.031ug / ml, 0.523ug / ml, 0.367ug / ml, 0.281ug / ml, 0.297ug / ml, 0.281ug / ml, 0.289ug / ml, 0.305ug / ml, 0.297ug / ml, and 0.289ug / ml.

7. Use of the photosensitizer according to claim 2 in the preparation of an inhibitor for inhibiting Microsporum canis, characterized in that: The MIC mean values ​​of the wormwood extract under different red light irradiation energy groups: 70W / J, 80W / J, 90W / J, 100W / J, 110W / J, 120W / J, 130W / J, 140W / J, 150W / J irradiation conditions are: 32mL / 100mL, 32mL / 100mL, 16mL / 100mL, 16mL / 100mL, 16mL / 100mL, 8mL / 100mL, 8mL / 100mL, 32mL / 100mL, 32mL / 100mL; the Eclipta prostrata extract The mean MIC values ​​under different red light irradiation energy groups: 60W / J, 70W / J, 80W / J, 90W / J, 100W / J, 110W / J, 120W / J, 130W / J, 140W / J, and 150W / J were 32mL / 100mL, 32mL / 100mL, 16mL / 100mL, 16mL / 100mL, 16mL / 100mL, 8mL / 100mL, 8mL / 100mL, 4mL / 100mL, 16mL / 100mL, and 16mL / 100mL, respectively.

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