Use of bakuchiol in the preparation of a medicament for treating vitiligo and a system for treating vitiligo

Abstract

The application provides application of bakuchiol in preparation of a medicine for treating vitiligo and a system for treating vitiligo, and belongs to the technical field of medicines. The application provides application of bakuchiol in preparation of a medicine for treating vitiligo, and the application adopts flavonoid compound bakuchiol to prepare the medicine for treating vitiligo, has smaller toxicity, and has a good curative effect on vitiligo in combination with ultraviolet light (such as UVA).

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to the application of psoralen in the preparation of drugs for treating vitiligo and a system for treating vitiligo. Background Technology

[0002] Vitiligo is an incurable acquired autoimmune disease characterized by white or reduced-pigmentation patches on the skin, caused by the destruction or loss of function of melanocytes in the skin. Melanocytes are responsible for producing melanin, a pigment that gives color to the skin, hair, and eyes. Vitiligo can occur anywhere on the body, but is most common on the face, hands, and feet. While vitiligo is not life-threatening, it can significantly reduce a patient's quality of life. Vitiligo is sometimes associated with other autoimmune diseases, such as thyroid disease, diabetes, and alopecia areata, but the exact cause is not fully understood.

[0003] Currently, treatments can help vitiligo patients control the progression of the disease and may restore pigmentation in some affected skin areas, slowing or stopping the destruction of melanocytes and improving the appearance of the skin. Photodependent irradiation (PUVA) therapy is a recognized symptomatic treatment for skin diseases such as psoriasis, atopic dermatitis, vitiligo, and cutaneous T-cell lymphoma. Currently, PUVA therapy for vitiligo typically uses coumarin compounds such as psoralen (PSO, CAS number 66-97-7) or its derivatives 8-methoxypsoralen (8-MOP, CAS number 298-81-7), 5-methoxypsoralen (5-MOP, CAS number 484-20-8), and 4,5',8-trimethylpsoralen (TMP, CAS number 3902-71-4) as active agents. During treatment, patients take these active agents orally, and then the affected skin areas are irradiated with UVA (ultraviolet light with wavelengths between 315 and 400 nm). However, the use of the aforementioned coumarin compounds as surfactants has significant toxicity. Summary of the Invention

[0004] The purpose of this invention is to provide the application of psoralen in the preparation of drugs for treating vitiligo and a system for treating vitiligo. This invention uses psoralen as a therapeutic drug in combination with ultraviolet light (such as UVA) to treat vitiligo, with low toxicity.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides the application of psoralen in the preparation of a drug for treating vitiligo.

[0007] Preferably, the drug comprises psoralen and a pharmaceutically acceptable carrier.

[0008] Preferably, the content of psoralen in the drug is 0.1-10 wt%.

[0009] Preferably, the dosage form of the drug includes a tincture.

[0010] Preferably, the method of administration of the drug includes topical application.

[0011] Preferably, the vitiligo includes vitiligo induced by monobenzone cream.

[0012] This invention provides a system for treating vitiligo, comprising an ultraviolet light source and a chemical drug, wherein the chemical drug includes psoralen.

[0013] Preferably, the ultraviolet light emitted by the ultraviolet light source has a wavelength range of 315–400 nm.

[0014] Preferably, the wavelength of the ultraviolet light is 365 nm.

[0015] This invention provides the application of psoralen in the preparation of drugs for treating vitiligo. This invention uses the flavonoid compound psoralen to prepare drugs for treating vitiligo, which have low toxicity and have a good therapeutic effect on vitiligo when combined with ultraviolet light (such as UVA). Attached Figure Description

[0016] Figure 1 Comparison of liquid chromatography images of psoralen and adenine before and after light exposure;

[0017] Figure 2 Comparison of liquid chromatography images of psoralen and guanine before and after light exposure;

[0018] Figure 3 Comparison of liquid chromatography images of psoralen and thymine before and after light exposure;

[0019] Figure 4 Comparison of liquid chromatography images of psoralen and cytosine before and after light exposure;

[0020] Figure 5 The liquid chromatogram of psoralen photoreaction on thymine / AT-DNA;

[0021] Figure 6 The image shows the results of LC-MS analysis of the photoaddition of psoralen to thymine;

[0022] Figure 7 The diagram shows the inferred structure and fragmentation pattern of the photoadduct;

[0023] Figure 8 The graph shows the color changes of psoralen and AT-DNA solutions after different light exposure times.

[0024] Figure 9 The ultraviolet absorption spectra of psoralen and thymine under thymine overdose conditions at different light exposure times;

[0025] Figure 10 The ultraviolet absorption spectra of psoralen and thymine under conditions of psoralen overdose were obtained after different exposure times.

[0026] Figure 11 The ultraviolet absorption spectrum of AT-DNA was obtained by gradually adding AT-DNA to a certain concentration of psoralen.

[0027] Figure 12 Comparison of the ultraviolet absorption spectra of psoralen, AT-DNA, and photoadducts;

[0028] Figure 13 The infrared absorption spectrum of psoralen on the photoreaction of thymine;

[0029] Figure 14 The femtosecond transient absorption spectrum of psoralen after excitation at 340 nm;

[0030] Figure 15 The femtosecond transient absorption spectrum of the BA-DNA adduct after excitation at 340 nm;

[0031] Figure 16 The one-dimensional and two-dimensional conductance signal diagrams of BA;

[0032] Figure 17 One-dimensional and two-dimensional conductivity signal diagrams of AT-DNA;

[0033] Figure 18 The diagram shows the one-dimensional and two-dimensional conductance signals of ATUV;

[0034] Figure 19 The diagrams show the one-dimensional and two-dimensional conductance signals of BAAT.

[0035] Figure 20 The diagram shows the one-dimensional and two-dimensional conductance signals of BAATUV;

[0036] Figure 21 The length of the uplifted compound;

[0037] Figure 22 The image shows the test results of the therapeutic effect of psoralen on the progression of vitiligo.

[0038] Figure 23 The image shows the results of the anti-inflammatory ability test of psoralen.

[0039] Figure 24 Figure showing the effects of psoralen on oxidative stress and CD8 T cell infiltration in mice;

[0040] Figure 25 The results of toxicity tests for psoralen methyl methacrylate (PMMA) in the treatment of vitiligo;

[0041] Figure 26 This is a schematic diagram illustrating the mechanism of action of psoralen methyl methacrylate (PMMA) in treating vitiligo according to the present invention. Detailed Implementation

[0042] This invention provides the application of psoralen in the preparation of a drug for treating vitiligo.

[0043] The CAS number of the psoralen methylphenidate described in this invention is 19879-32-4, and its structural formula is shown in Formula I:

[0044]

[0045] Currently, PUVA therapy for vitiligo typically uses coumarin compounds or their derivatives as active agents, such as psoralen (CAS No. 66-97-7, structural formula as shown in Formula A), 8-methoxypsoralen (CAS No. 298-81-7, structural formula as shown in Formula B), 5-methoxypsoralen (CAS No. 484-20-8, structural formula as shown in Formula C), or 4,5',8-trimethylpsoralen (CAS No. 3902-71-4, structural formula as shown in Formula D). However, the use of these coumarin compounds as active agents is relatively toxic and may cause adverse reactions such as nausea, vomiting, stomach discomfort, headache, insomnia, nervousness, nausea, vomiting, and depression. Research on flavonoids is limited, and there is currently no research on the role of psoralen in the prevention and treatment of vitiligo.

[0046]

[0047] This invention uses the flavonoid compound psoralen to prepare a drug for treating vitiligo. It has low toxicity and, when combined with ultraviolet light (such as UVA), has a good therapeutic effect on vitiligo. Specifically, in this embodiment of the invention, liquid chromatography-mass spectrometry (LC-MS) is used to predict the possible binding modes of psoralen to DNA. Then, multispectral methods, including ultraviolet-visible (UV-vis) absorption spectroscopy, infrared (IR) absorption spectroscopy, femtosecond transient absorption spectroscopy (fs-TA), ultra-high performance liquid chromatography (UPLC), and STM-BJ are used to determine the interaction between psoralen and DNA. Among these methods, LC-MS and UHPLC analysis can provide molecular weight and rich structural information for each component, making the qualitative analysis results more reliable and faster. Femtosecond transient absorption spectroscopy can perform time-resolved data analysis, which is suitable for complex photophysical and photochemical processes. STM-BJ technology can achieve precise measurement of the conductivity of individual molecules, which is crucial for understanding the charge transport mechanism when molecules interact with DNA. This technology can rapidly construct thousands of single-molecule junctions in a short time, capture the dynamic changes of individual molecules, and present them in the form of electrical signals, with good repeatability and high temporal accuracy. Furthermore, based on the established ability of psoralen to bind to thymine bases, this invention investigated the therapeutic effect of psoralen on vitiligo, revealing its potential mechanism for treating vitiligo and providing guidelines for the rational improvement of PUVA active agents. Moreover, the hepatotoxicity and nephrotoxicity of psoralen as an active agent used in this invention are lower than those of coumarin compounds currently used clinically.

[0048] In this embodiment of the invention, the drug may include psoralen and a pharmaceutically acceptable carrier; the invention does not specifically limit the type of the pharmaceutically acceptable carrier, and any pharmaceutically acceptable carrier known to those skilled in the art may be used; the content of psoralen in the drug may be 0.1-10 wt%, further may be 0.5-5 wt%, further may be 0.8-2 wt%, and even further may be 0.9-1.5 wt%.

[0049] In this embodiment of the invention, the dosage form of the drug specifically includes a tincture. When the dosage form of the drug is a tincture, the content of psoralen in the drug can be 9 mg / mL. The method of administration of the drug specifically includes topical application.

[0050] In this embodiment of the invention, the efficacy of psoralen in treating vitiligo was specifically verified by using monobenzone cream-induced vitiligo.

[0051] This invention provides a system for treating vitiligo, comprising an ultraviolet light source and a chemical drug, wherein the chemical drug is a drug including psoralen. In embodiments of this invention, the chemical drug may be the drug for treating vitiligo described in the above-described technical solution, and will not be elaborated further here.

[0052] In this embodiment of the invention, the ultraviolet light emitted by the ultraviolet light source can be in the wavelength range of 315-400nm, and the wavelength of the ultraviolet light can specifically be 315nm, 330nm, 345nm, 360nm, 365nm, 370nm, 385nm or 400nm; this embodiment of the invention specifically uses an ultraviolet light wavelength of 365nm as an example to verify the therapeutic effect of psoralen combined with ultraviolet light on vitiligo.

[0053] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0054] The following are some of the materials used in the following embodiments:

[0055] Psoralen (BA) was purchased from Maclean's (Shanghai, China, ≥98%), and adenine, thymine, cytosine, and guanine were purchased from Solarbio (Beijing, China, ≥98%). Lyophilized oligonucleotide 5'-(TA) 20 -3' was purchased from Sigma-Aldrich. The manufacturer purified the sample using HPLC. The oligonucleotide chains in the solution were annealed within 24 hours prior to measurement by heating the solution to 93°C in a water bath and cooling it to room temperature over several hours. The lyophilized DNA powder was then dissolved in ultrapure water to obtain the DNA solution. Other solvents used were ultrapure water (HPLC gradient grade), anhydrous ethanol (analytical grade), methanol (chromatographic grade), and acetonitrile (chromatographic grade).

[0056] In the following examples, all data used in the statistical analyses are expressed as mean ± standard error of at least three independent experiments. One-way ANOVA was used for comparisons among multiple groups, and Student's t-test (two-tailed) was used for comparisons between two groups. Statistical analyses were performed using GraphPadPrism software. A p-value <0.05 indicates statistical significance.

[0057] Example 1 UPLC Analysis

[0058] Analysis method:

[0059] Analysis was performed on a Waters ACQUITY UPLC system controlled by Empower Pro software (Waters). An XBridge Premier Oligonucleotide BEH C18 column (4.6 × 150 mm, 2.5 μm) was used, with gradient elution at 30 °C. The mobile phase consisted of mobile phase B (acetonitrile) and mobile phase D (0.1% formic acid aqueous solution). The gradient elution program was as follows: 0–10.67 min, the volume fraction of mobile phase B increased uniformly from 5% to 50%; 10.67–19.67 min, the volume fraction of mobile phase B increased uniformly from 50% to 95%; 19.67–23.42 min, the volume fraction of mobile phase B decreased uniformly from 95% to 5%. The detection wavelength was 254 nm. The mobile phase flow rate was 1.2 mL / min, and the injection volume was 5 μL.

[0060] Results and Discussion:

[0061] In this embodiment, the photoaddition reaction of psoralen with four bases—adenine (A), guanine (G), thymine (T), and cytosine (C)—was analyzed by ultra-high performance liquid chromatography. Figure 1 This is a comparison of liquid chromatography (LC) images of psoralen and adenine before and after light exposure. Figure 2 This is a comparison of liquid chromatography (LC) images of psoralen and guanine before and after light exposure. Figure 3 This is a comparison of liquid chromatography (LC) images of psoralen and thymine before and after light exposure. Figure 4 This is a comparison of the liquid chromatography (LC) chromatograms of psoralen and cytosine before and after light exposure. Orange represents the LC chromatogram before light exposure, and blue represents the LC chromatogram after light exposure. The results show that no new compound peaks were generated in the mixed solution of psoralen and A and G bases before and after light exposure (e.g., Figure 1 and Figure 2 (As shown); however, a mixed solution of psoralen and T and C bases produced a new peak after a certain period of light exposure, and this peak was located between the peaks of psoralen and thymine. It is speculated that the new peak is due to the formation of a photoadduct (such as...). Figure 3 and Figure 4 (As shown). However, the peak area of ​​the photoadduct bound to thymine is larger than that bound to cytosine, indicating that psoralen can bind to DNA and preferentially bind to thymine to form a photoadduct.

[0062] Based on the confirmed ability to bind with thymine, this embodiment then analyzed the liquid chromatograms of psoralen, thymine, and AT-DNA after different light exposure times. Figure 5 The liquid chromatogram of psoralen photoreaction on thymine / AT-DNA; Figure 5 Image A shows the liquid chromatograms of psoralen and thymine after different light exposure times. Figure 5 Figure B shows the liquid chromatograms of psoralen under different light exposure times. Figure 5 C represents the liquid chromatograms of psoralen and AT-DNA after different light exposure times. Figure 5 Figure D shows a comparison of liquid chromatography (LC) chromatograms of different proportions of psoralen and thymine, and psoralen after 96 hours of light exposure. Peaks 1 and 2 represent thymine, peak 4 represents psoralen, and peak 3 represents the photoadduct. The results show that with increasing light exposure time, peak 4 gradually decreases, while peak 3 gradually increases, reaching its maximum at 96 hours of light exposure (e.g., [image missing]). Figure 5 (As shown in Figure A). Replace thymine with AT-DNA (e.g., Figure 5 (As shown in C) Results and Figure 5 The values ​​in A remain consistent. Liquid chromatograms of psoralen alone exposed to light for different durations are used as a control (e.g., ...). Figure 5 (As shown in Figure B). To improve the yield of photoadduct formation, the amounts of different ratios of psoralen and thymine forming photoadducts were screened (e.g., ...). Figure 5 As shown in Figure D), the results showed that after 96 hours of illumination, the peak area of ​​peak 3 was larger when the mass ratio of psoralen to thymine was 1:10 or 1:20, indicating a higher yield of photoadduct.

[0063] Example 2 LC-MS Analysis

[0064] Analysis method:

[0065] Samples were analyzed using a Dionex Ultimate 3000 HPLC system (Thermo Fisher Scientific, Waltham, MA, USA) and a Thermo Scientific Q Exactive system. For LC-MS / MS analysis, compounds were ionized using electrospray ionization in both positive and negative modes under the following source conditions: HESI ion source, sheath gas rate of 40 arb, auxiliary gas rate of 10 arb, spray voltage of 3.8 kV for positive ions and 3.0 kV for negative ions, capillary temperature of 320 °C, auxiliary gas temperature of 300 °C, scan mode of Fullms / dd-ms2top10, and collision voltages of NCE 15, 30, and 45. Chromatographic separation was achieved by injecting 5 μL of the sample at a flow rate of 0.5 mL / min onto an Eclipse Plus C18 column (100 mm × 4.6 mm, 3.5 μm). The mobile phase consisted of mobile phase A, which was a 0.1% (v / v) aqueous solution of formic acid, and mobile phase B, which was acetonitrile. Gradient elution was employed, with the following elution program: 0–13 min, the volume fraction of mobile phase B was increased uniformly from 5% to 50%; 13–25 min, the volume fraction of mobile phase B was increased uniformly from 50% to 95%; 25.1–30 min, the volume fraction of mobile phase B was decreased uniformly from 95% to 5%.

[0066] Results and Discussion:

[0067] In this embodiment, the photobinding of psoralen and thymine was further analyzed by LC-MS. Figure 6 The results of the photoaddition of psoralen to thymine by LC-MS are shown in Figure A, where A represents the total negative ion chromatogram of psoralen and thymine before and after irradiation; B represents the total positive ion chromatogram of psoralen and thymine before and after irradiation; C represents the fragmentation information of psoralen; D represents the fragmentation information of thymine; E represents the retention time of the photoadduct in secondary mass spectrometry; and F represents the fragmentation information of the photoadduct. The results show that after 96 hours of irradiation, the absorption intensity of psoralen decreases, and new peaks are generated (e.g., [missing information]). Figure 6 (As shown in A and B). Figure 6 C and D represent the fragmentation information of psoralen and thymine, respectively. A search of secondary mass spectrometry for thymine (relative molecular mass 127.0506) revealed a compound with a relative molecular mass of 451.1874 at 12.65 min. The fragmentation information for this compound included fragments of both psoralen and thymine (e.g.,...). Figure 6 (As shown in E and F), this indicates that psoralen can react with thymine to form a photoadduct upon light excitation.

[0068] Figure 7 The diagram shows the predicted structure and fragmentation pattern of the photoadduct. The results indicate that under light excitation, the isopentenyl double bond of psoralen and the carbon-carbon double bonds at positions 5 and 6 of the thymine base break and then combine to form a cyclobutanepyrimidine dimer.

[0069] Example 3 Spectral Characteristics

[0070] 1. Ultraviolet-visible (UV-vis) absorption spectrum and infrared (IR) spectrum

[0071] Analysis method:

[0072] UV-Vis absorption spectra were obtained using a UV-2600. The solutions were measured in fused silica cuvettes (1 cm path length) from Hellma. In the UV / Vis experiments, the effect of photoaddition on the UV absorption spectra was investigated by irradiating the sample solutions at 365 nm with a xenon lamp. The solutions were irradiated in beakers under magnetic stirring. IR spectroscopy is commonly used to characterize the specific binding properties of small molecule-DNA interactions and to monitor the influence of small molecules on DNA structure. Infrared spectra were measured using a Bruker Vertex 70V infrared spectrometer. Measurements were performed at room temperature (20–21 °C) using a custom cuvette with a CaF2 window (100 μm path length).

[0073] Results and Discussion:

[0074] 1.1 Photoaddition-induced UV-Vis absorption characteristics

[0075] Ultraviolet spectroscopy is one of the most commonly used methods for detecting the interaction between small molecules and DNA. Figure 8 The image shows the color changes of the solution of psoralen and AT-DNA after different light exposure times. The results show that after the mixture of psoralen and thymine is exposed to light for a longer period of time, the solution color gradually deepens until it turns dark yellow.

[0076] Figure 9 The UV absorption spectra of psoralen and thymine under thymine excess conditions and illumination for different durations are shown. The results indicate that under thymine excess conditions, UV absorption is dominated by thymine absorption, with two absorption bands around 215 nm and 263 nm. With increasing illumination time, the absorption intensity of thymine at 215 nm and 263 nm decreases, exhibiting blue shift and red shift, respectively. The absorption intensity between 350 and 425 nm gradually increases, which was not present before illumination.

[0077] Figure 10The UV absorption spectra of psoralen and thymine under excess illumination for different durations were obtained. The results showed that under excess psoralen conditions, UV absorption was dominated by psoralen, exhibiting three absorption bands around 220 nm, 277 nm, and 325 nm. With increasing illumination time, the absorption of psoralen at 220 nm, 277 nm, and 325 nm became more gradual, a phenomenon not present before illumination.

[0078] Figure 11 This study investigated the UV absorption spectra of psoralen with gradually added AT-DNA at a specific concentration. To further verify the photoaddition reaction between the compounds, the UV absorption spectra of psoralen were studied in the absence and presence of different concentrations of DNA. The results showed that with increasing DNA content, the absorption intensity of psoralen decreased at 220 nm, 277 nm, and 325 nm, and increased between 350 and 425 nm.

[0079] Figure 12 The image shows a comparison of the UV absorption spectra of psoralen, AT-DNA, and photoadducts. The results show that the UV absorption spectra of the three are significantly different.

[0080] The above results strongly demonstrate that psoralen can interact with DNA and form a photoadduct (i.e., the BA-DNA complex).

[0081] 1.2 Infrared absorption characteristics of photoaddition

[0082] IR spectroscopy is often used to characterize the specific binding properties of small molecule-DNA interactions and to monitor the effects of small molecules on DNA structure. Figure 13 The infrared absorption spectrum of psoralen on the photoreaction of thymine; Figure 13 A in the image represents the IR spectrum of psoralen. Figure 13 B represents the IR spectrum of thymine. Figure 13 C represents the IR spectra of psoralen and thymine after different illumination times. Figure 13 In the middle D section, we see the IR contrast diagrams of psoralen, thymine, and their photoadducts. (See diagram for example.) Figure 13 As shown in Figures A and B, the spectrum of psoralen is at 1714 cm⁻¹. -1 There is a vibrational band at 1651 cm, which is attributed to the carbonyl stretching vibration; -1 A broadband cable of 1579cm is located at [location missing]. -1 A broadband band at this point is attributed to the C=C stretching vibration. Thymine has three distinct frequency bands in the high-frequency range. 1731cm -1 and 1674cm -1The band at 1600 cm⁻¹ can be assigned to the carbonyl stretching vibration. -1 The wavelength band at that point can be assigned to the C=C stretching vibration. In a buffer solution of psoralen and thymine, approximately 95% of the psoralen is inserted. The initial spectrum of BA-T before illumination is primarily dominated by thymine vibrations. Figure 13 As shown in C and D, illumination with a xenon lamp source system emitting light at 365 nm causes a change in the infrared spectrum. (1700 cm⁻¹) -1 The changes in absorption around the left and right may be due to the carbonyl stretching vibration of psoralen and the bleaching effect of thymine. 1640cm -1 The changes in absorption on the left and right sides may be due to the vibration of thymine. 500cm -1 The absorption intensity in nearby wavelengths increases with prolonged illumination time and shows a slight shift towards higher wavenumbers. This temporal behavior suggests that it is due to secondary photochemistry, specifically the formation of crosslinks.

[0083] 2. Femtosecond transient absorption spectrum

[0084] Analysis method:

[0085] Femtosecond transient absorption (fs-TA) data were acquired using the Pharos transient absorption spectrum harpia femtosecond laser, with the Pharos laser system providing the base source. Probes were obtained by utilizing changes in pump absorbance, and the probe beam was set to a magic angle. The time resolution was 180 fs. The excitation wavelength was 340 nm, and the pump power was 1 μJ / cm². 2 The instrument employs a detection range of 370–500 nm, a repetition frequency of 5 Hz, and a pulse duration of 8 ns for photoexcitation. The instrument's time-zero offset is determined and corrected using the optical Kerr effect as a function of wavelength. Signals from the pure solvent are recorded in separate measurements and subtracted after appropriate scaling to eliminate time-zero artifacts. The solution is circulated through a fused silica flow cell with a path length of 1 mm. The sample volume is large enough that the contribution of photoproducts formed during the experiment is negligible.

[0086] Results and Discussion:

[0087] A BA solution containing AT-DNA was excited using a UVA laser pulse, and detection was performed in the UV / Vis region. The BA and AT-DNA solutions were placed in a buffer solution and excited with a 340 nm laser pulse. Absorption below 340 nm could not be obtained for the selected excitation wavelength. Figure 14 The femtosecond transient absorption spectrum of psoralen after excitation at 340 nm is shown. Figure 15The image shows the femtosecond transient absorption spectrum of the BA-DNA adduct after excitation at 340 nm. The results show that near time zero, the BA solution exhibits a significant positive absorption in the 390–470 nm range, peaking at 430 nm, while a significant negative absorption occurs at 373 nm (e.g., [missing information]). Figure 14 (As shown). Adding AT-DNA to BA solution alters the decay kinetics of BA (e.g., Figure 15 As shown in the figure, the positive absorption in the 390–470 nm range and the negative absorption at 373 nm are significantly reduced, while the positive absorption below 390 nm and above 480 nm increases. The kinetic curve and SPE spectrum also show the same result. The above results indicate the formation of photoadducts.

[0088] 3. Conductivity Measurement

[0089] Analysis method:

[0090] The conductivity of molecular junctions was measured using a Scanning Tunneling Microscope-Break Junction (STM-BJ) instrument. BAATUV compounds were generated by repeatedly forming and breaking atomic contacts between a gold tip and a gold substrate with 99.99% purity. Repeated on-off cycles were performed at room temperature in a mixed solution containing 1 μM of the target molecule, psoralen, and DNA, with a 100 mV bias applied between the gold tip and the gold substrate. The current-to-voltage converter used for data acquisition operated at 50 kHz. Throughout all break junction measurements in this embodiment, the sample was immersed in the solvent, and both the gold tip and the gold substrate remained immersed in the solvent at the end of all measurements.

[0091] Results and Discussion:

[0092] For conductivity measurements, a solution of the target molecule was dropped onto a gold substrate, while a gold STM-BJ probe, suspended atop the substrate, was brought close to the substrate by a stepper motor and a piezoelectric stack, forming an Au-Au point contact. Molecules bonded to the gold tip and substrate, forming stacked supramolecular junctions. All molecules were dissolved in PBS buffer at a concentration of 1 μM. Conductivity measurements were performed at room temperature with a 0.1 V bias applied between the gold tip and substrate. The flow cell was illuminated with a 365 nm UV light source, and conductivity traces obtained during the break-bond measurements of each molecule were collected to construct one-dimensional and two-dimensional conductivity signal maps.

[0093] Figure 16 The diagrams show the one-dimensional and two-dimensional conductance signals of BA. Figure 17 The diagrams show the one-dimensional and two-dimensional conductance signals of AT-DNA. Figure 18 The diagrams show the one-dimensional and two-dimensional conductance signals of ATUV. Figure 19The diagrams show the one-dimensional and two-dimensional conductance signals of BAAT. Figure 20 The diagram shows the one-dimensional and two-dimensional conductance signals of BAATUV. Figure 21 The molecular length of the uplift compound is given. A t-test was used for comparisons between two groups, and a one-way ANOVA was used for comparisons between multiple groups; *p<0.05, **p<0.01, ***p<0.001. The results showed that the one-dimensional and two-dimensional conductance signal intensities of BA, AT, ATUV, and BAAT were within 10... -4 ~10 -2 Between (e.g.) Figures 16-19 As shown), the conductivity signal of the BA and AT-DNA mixed solution is significantly enhanced under light excitation (e.g., Figure 20 As shown in the figure, the enhancement of the conductivity signal indicates that BA binds to AT-DNA to produce a photoadduct under light excitation. The molecular lengths stretched in each group were then statistically analyzed, as shown in the figure. Figure 21 As shown, the molecular length of BA and AT-DNA increased significantly after light exposure. This significant increase in molecular length indicates that BA binding increases the length of AT-DNA through insertion.

[0094] Example 4 Animal Experiment

[0095] Analysis method:

[0096] 1. Establishment of a mouse model of vitiligo and drug administration regimen

[0097] Six-week-old male C57BL / 6 mice were purchased from Henan Sk贝斯 Biotechnology Co., Ltd. (SCXK(Yu)2020-0005). Animal ethics was approved by the Ethics Committee of the First Affiliated Hospital of Shihezi University. The animals were housed in a temperature-controlled environment maintained at 20-25 °C and subjected to a standard 12-h light and 12-h dark cycle. Throughout the experiment, the mice had unrestricted access to food and water. All mice were acclimated for 7 days before the start of the experiment. Before the start of the experiment, the hair on the backs of a total of 70 C57BL / 6 mice was shaved off with an electric razor to cover an area of 2 cm × 2 cm. In all groups except the control group, 40% monobenzone cream was applied to the depilated area once a day, and an equal amount of sterile water was applied to the depilated area in the control group. An experimental vitiligo mouse model was established for 20 consecutive days. After the modeling was completed, the mice were divided into a model group, an 8-MOP group, an Et-OH group, a UV group, a BA group, and a BAUV (i.e., psoralen + light) group, with 10 mice in each group. From the 21st day of modeling, drug administration was applied by smearing once a day for 28 consecutive days, 1 mL each time. Subsequently, the skin lesions were scored using the bleaching scoring method, and skin tissues and sera were collected on the last day of treatment for further analysis. A scoring system based on the degree of depigmentation in the treatment area was used to evaluate the vitiligo model. A score of 1 indicates that the depigmented area is less than 10%, a score of 2 indicates that the depigmented area is less than 10-25%, a score of 3 indicates that the depigmented area is below 25-50%, a score of 4 indicates that the depigmented area is below 50-75%, and a score of 5 indicates that the depigmented area exceeds 75%. White hair counting was performed by visually examining the back area of each mouse and calculating the number of white hair clusters present using Image J. These methods were used to evaluate the effectiveness of vitiligo induction and to quantify the degree of depigmentation and the appearance of white hair in experimental mice.

[0098] 2. Histological analysis

[0099] The skin tissues of the depigmented area were fixed in � wt% paraformaldehyde buffer, dehydrated with a series of ethanol (75%, 85%, 95% and 100%) and xylene, and embedded in paraffin. Then the processed tissues were cut into 5-μm sections. These sections were stained with HE, Masson and Masson-Fontana to accurately determine the melanin content. These staining protocols laid a reliable foundation for further exploring the tissue structure and were beneficial for the preliminary evaluation of the melanin content and related structures.

[0100] 3. Evaluation of antioxidant stress and anti-inflammatory activities in the skin tissues of vitiligo mice

[0101] Following BAVACHIN treatment, mouse skin tissue was collected, and tissue homogenates were obtained using the extraction buffer provided with the kit. The supernatant was then collected. Subsequently, the activities of superoxide dismutase (SOD) and catalase (CAT), as well as the concentration of malondialdehyde (MDA), were quantified, and antioxidant stress levels were assessed using commercial assay kits. CAT and SOD activities were measured using an SOD activity assay kit (JL-T0781, Jianglai Biotechnology) and a CAT activity assay kit (JL-T0900, Jianglai Biotechnology), respectively. MDA concentration was measured using an MDA content assay kit (JL-T0761, Jianglai Biotechnology) according to the manufacturer's protocol. Simultaneously, mouse blood was collected by centrifugation (4℃, 3000 rpm, 15 min), followed by serum collection. Subsequently, following the manufacturer's recommended procedure, commercial ELISA kits purchased from U-Bio Biotechnology were used to measure the levels of tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), and interleukin-6 (IL-6) in mouse serum to assess their anti-inflammatory activity.

[0102] 4. Immunofluorescence staining

[0103] To perform immunofluorescence analysis on the skin tissue, a small patch of skin was removed from the destaining area on the back of a mouse. The skin tissue was fixed in 4 wt% paraformaldehyde at 4 °C for 24 h, and then dehydrated in 30 wt% sucrose solution at 4 °C. Subsequently, the tissue was frozen in the optimal cutting temperature (OCT) compound and cut into 15 μm sections using a cryostat (RWD FS800A, Shenzhen, China). The sections were washed three times with PBS to remove OCT, and then immunostained with Alexa Fluor 647CD8a (100724, BioLegend, San Diego, CA, USA, 1:200) to detect CD8 T cells in the skin tissue. In addition, immunostaining was performed using TNF-α polyclonal antibody (17590-1-AP, Proteintech, Chicago, IL, USA, 1:200), IFN Gamma polyclonal antibody (15365-1-AP, Proteintech, Chicago, IL, USA, 1:200), and Goat AntiRabbit IgG (H&L)-Alexa Fluor 594 (RS3611, Immunoway, Plano, TX, USA, 1:200) to assess the levels of inflammatory factors in skin tissue. All sections were imaged using laser scanning confocal microscopy. Finally, the acquired data were analyzed using ImageJ software to provide quantitative information on CD8 T cell-related fluorescence signals and inflammatory factors.

[0104] Results and Discussion:

[0105] 1. The intervention effect of BAUV on the progression of vitiligo

[0106] This embodiment uses a vitiligo mouse model induced by 4% monobenzone cream to study the potential of BA intervention in vitiligo progression. Figure 22 The figures show the therapeutic effects of psoralen on the progression of vitiligo. A represents the animal model and treatment diagram; B shows the depigmentation results on the backs of mice, with white arrows indicating white hair areas; C shows the pigment loss score of vitiligo mice; D shows a statistical graph of the number of white hairs after treatment, with data expressed as standard error ± mean (n=6); E shows the HE staining, Masson staining, and Masson-Fontana staining results of mouse skin tissue, with representative images shown and a scale bar of 200 μm. The t-test was used for comparisons between two groups, and one-way ANOVA was used for multiple groups; *p<0.05, **p<0.01, ***p<0.001.

[0107] The results showed that after establishing the vitiligo model (day 0), mice in each treatment group received treatment and were sacrificed four weeks later (e.g., Figure 22 (As shown in Figure A). The degree of depigmentation of the back hair of mice was assessed by visual observation. Compared with the normal group, the back hair of mice showed significant pigment loss after application of monobenzone cream to the designated area. However, after intervention, reduced pigment loss was observed in the BAUV group, a phenomenon further confirmed by statistical analysis (e.g., Figure 22 (As shown in Figure B). Statistical analysis of depigmentation scores after mouse modeling showed that the depigmentation score was significantly increased in the vitiligo group, while a significant decrease was observed after BAUV (vitiligo + BAUV) treatment, highlighting the significant difference between the two groups (e.g., ...). Figure 22 (As shown in C). Furthermore, this embodiment calculated the number of white hairs in the normal group, vitiligo group, and each treatment group. The results showed that the number of white hairs on the backs of mice in the vitiligo + BAUV group was significantly less than that in the vitiligo group (e.g., as shown in C). Figure 22 (As shown in Figure D). To assess the histological changes in skin tissue, Masson-Fontana staining and HE staining were performed in this embodiment to examine the dorsal skin of vitiligo mice, particularly changes in melanin granules. HE staining results showed that the skin of the vitiligo group mice was significantly lacking in hair follicles, accompanied by mild spongiform changes and basal cell vacuolation. In contrast, the skin showed significant improvement after treatment, characterized by an increase in hair follicles and a reduction in pathological changes. In Masson-Fontana staining, melanin underwent a silvery reaction, leading to the formation of melanin granules. After four weeks of intervention, the amount of melanin around the hair follicles in the treatment group was significantly increased. In addition, the number of melanin granules migrating to the skin surface was significantly increased (e.g., ...). Figure 22(As shown in E). These findings collectively suggest that BAUV has the potential to intervene in the progression of vitiligo in mice, as evidenced by the reduced depigmentation and favorable histological changes observed in the treatment group.

[0108] 2. Anti-inflammatory effect of BAUV on vitiligo mice

[0109] Inflammatory factor levels are key indicators for assessing vitiligo patients. Therefore, measuring inflammatory factor levels is a common method for evaluating the progress and effectiveness of vitiligo treatment. Figure 23 The figures show the results of the anti-inflammatory activity test of psoralen. A, B, and C represent the serum TNF-α, IFN-γ, and IL-6 concentrations in mice treated with psoralen for 28 days, respectively. D and E are immunofluorescence images of skin inflammatory factors in different groups of mice (blue: DAPI; red: TNF-α, IFN-γ). The scale bar is 100 μm. The t-test was used for comparisons between two groups, and one-way ANOVA was used for comparisons between multiple groups; *p<0.05, **p<0.01, ***p<0.001.

[0110] The results showed that monobenzone-induced vitiligo mice had significantly increased serum levels of pro-inflammatory cytokines (such as TNF-α, IFN-γ, and IL-6). However, after BAUV treatment, the levels of these inflammatory factors significantly decreased (e.g., Figure 23 (As shown in A, B, and C). Furthermore, in this embodiment, immunofluorescence staining was performed on the skin of the depigmented areas of mice. The results showed that the expression of inflammatory factors in the skin of the vitiligo group was significantly higher than that of the other two groups (e.g., ...). Figure 23 (As shown in D and E). These results highlight the effectiveness of BAUV intervention in suppressing inflammatory responses in vivo.

[0111] 3. Effects of BAUV on oxidative stress and CD8 T cell infiltration in vitiligo mice

[0112] Figure 24 The figures show the effects of psoralen on oxidative stress and CD8 T cell infiltration in mice. A, B, and C represent the CAT activity, SOD activity, and MDA content in mouse skin tissue after 28 days of psoralen treatment, respectively. D shows the immunofluorescence image of skin CD8 T cells (blue: DAPI; red: CD8+ T cells) after 28 days of psoralen treatment. The scale bar is 50 μm. T-tests were used for comparisons between two groups, and one-way ANOVA was used for multiple groups; *p<0.05, **p<0.01, ***p<0.001.

[0113] This embodiment evaluated the concentration of malondialdehyde (MDA) and the activities of superoxide dismutase (SOD) and catalase (CAT) in mouse skin tissue after intervention. Figure 24As shown in Figures A, B, and C, oxidative stress in the skin led to a sharp decrease in SOD and CAT levels and a significant increase in MDA levels in the vitiligo group. However, after BAUV treatment, MDA concentration significantly decreased, and SOD and CAT activities recovered (p<0.05). During the development of vitiligo, CD8 T cells are enriched in the dermis, leading to the destruction of melanocytes and potentially affecting melanin production, which is one of the possible factors in the pathogenesis of vitiligo. To explore the relationship between BAUV and immune infiltration, this study conducted an in-depth investigation of CD8 T cell infiltration, specifically by performing immunofluorescence staining on the depigmented skin areas to assess CD8 T cell infiltration. Figure 24 As shown in Figure D, the number of CD8 T cells in the skin of the vitiligo group was greater than that in the other two groups.

[0114] 4. Toxicity of BAUV in Vitiligo Mice

[0115] Figure 25 The results of toxicity tests on psoralen for the treatment of vitiligo are shown in Figure A, which contains photographs of the liver, spleen, and kidney of mice; Figures B, C, and D show the organ coefficients of the liver, kidney, and spleen of mice, respectively; and Figure E shows the HE staining results of the liver, spleen, and kidney of mice. The t-test was used for comparisons between two groups, and one-way ANOVA was used for comparisons between multiple groups; *p<0.05, **p<0.01, ***p<0.001.

[0116] In this embodiment, the organs of vitiligo mice after BAUV treatment were visually observed and evaluated, such as... Figure 25 As shown in Figure A, compared with the blank control group, the 8-MOP group, BA group, and BAUV group showed some degree of liver damage, while there were no significant differences in spleen and kidney function among the groups. Next, the organ coefficients of the mice in each group were statistically analyzed, as shown below. Figure 25 As shown in Figures B, C, and D, compared with the Control group, the liver weight of the 8-MOP, BA, and BAUV groups was significantly increased. The kidney weight of the 8-MOP and BA groups also showed some increase. There was no statistically significant difference in spleen weight among the treatment groups. To assess the histological changes in various organs and tissues, HE staining was performed in this example to examine organ changes in vitiligo mice. Figure 25As shown in Figure E, the liver tissue structure in the Control group was basically normal, with normal hepatocyte morphology, clear nuclei, abundant cytoplasm, and no edema or fatty degeneration. The hepatocytes were tightly and regularly arranged, with no obvious inflammatory cell infiltration, and no dilation of the hepatic sinusoids. The liver tissue structure in the 8-MOP group was severely abnormal, with a large number of inflammatory cells infiltrating the hepatic sinusoids, and some hepatocytes showing mild edema and cytoplasmic vacuolation. The liver tissue in the BA group was moderately abnormal, with some hepatic sinusoids showing mild dilation and cytoplasmic vacuolation. The liver tissue structure in the BAUV group was mildly abnormal, with hepatocytes tightly and regularly arranged, no obvious inflammatory cell infiltration, and some cytoplasmic vacuolation. In the Control group, the glomerular and tubular structures were normal, with no proliferation of mesangial cells and matrix, and no inflammatory cell infiltration or fibrosis in the renal interstitium. Some glomeruli in the 8-MOP and BA groups showed significant enlargement or shrinkage, while there was no significant difference in renal tissue between the BAUV and Control groups. No significant differences were observed in the spleens of each group except for the Model group. In the Model group, the junction between the red and white pulp was blurred, and no hyperplasia was observed in the spleen capsule and fibrous connective tissue of the splenic trabeculae, with no inflammatory cell infiltration.

[0117] Figure 26 This diagram illustrates the mechanism of action of psoralen methyl methacrylate (PMMA) in treating vitiligo. Specifically, PMMA effectively treats vitiligo by binding to the thymine bases of DNA. This mechanism involves regulating oxidative stress, restoring antioxidant capacity, and inhibiting inflammatory responses. PMMA treatment effectively reduces pigment loss, promotes melanin granule formation, and contributes to favorable histological changes in the skin of vitiligo mice.

[0118] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The use of psoralen as the sole active ingredient in the preparation of a drug for treating vitiligo, wherein the structural formula of psoralen is shown in Formula I: Formula I.

2. The application according to claim 1, characterized in that, The drug includes psoralen and a pharmaceutically acceptable carrier.

3. The application according to claim 2, characterized in that, The content of psoralen in the drug is 0.1~10wt%.

4. The application according to any one of claims 1 to 3, characterized in that, The dosage form of the drug includes tinctures.

5. The application according to claim 4, characterized in that, The drug can be administered via topical application.

6. The application according to claim 1, characterized in that, The vitiligo mentioned includes vitiligo induced by monobenzone cream.

Images