Application of psoralen A in preparation of medicine for treating vitiligo and system for treating vitiligo

Abstract

The invention provides application of psoralen A in preparation of a medicine for treating vitiligo and a system for treating vitiligo, and belongs to the technical field of medicines. According to the application of the psoralen A in preparing the medicine for treating the vitiligo, the medicine for treating the vitiligo is prepared by adopting the flavone compound psoralen A, the toxicity is low, and the medicine has a good curative effect on the vitiligo in combination with ultraviolet light (such as UVA).

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to application of psoralen in preparing a medicine for treating vitiligo and a system for treating vitiligo. Background Art

[0002] Vitiligo is an incurable acquired autoimmune disease characterized by white or hypopigmented patches on the skin due to the destruction or loss of function of melanocytes in the skin. Melanocytes are responsible for producing melanin, the pigment that gives skin, hair, and eyes their color. Vitiligo can occur anywhere on the body but is most common on the face, hands, and feet. While vitiligo is not directly life-threatening, it can reduce a person'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 pigment in some affected skin areas to slow or stop the destruction of melanocytes and improve the appearance of the skin. Photosensitive vasectomy (PUVA) therapy is a recognized symptomatic treatment for skin diseases, such as psoriasis, atopic eczema, vitiligo, and cutaneous T-cell lymphoma. Currently, PUVA therapy is used to treat vitiligo, usually using coumarin compounds such as psoralen (PSO, CAS No. 66-97-7) or its derivatives 8-methoxypsoralen (8-MOP, CAS No. 298-81-7), 5-methoxypsoralen (5-MOP, CAS No. 484-20-8), and 4,5',8-trimethylpsoralen (TMP, CAS No. 3902-71-4) as active agents. During the treatment, the patient takes the above active agents and then uses UVA (ultraviolet light with a wavelength between 315 and 400nm) to irradiate the relevant skin area. However, the above-mentioned coumarin compounds used as active agents are relatively toxic. Summary of the invention

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

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The invention provides an application of psoralen in preparing a medicine for treating vitiligo.

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

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

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

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

[0011] Preferably, the vitiligo comprises monobenzone cream-induced vitiligo.

[0012] The invention provides a system for treating vitiligo, comprising an ultraviolet light source and chemical drugs, wherein the chemical drugs are drugs including psoralen.

[0013] Preferably, the wavelength band of the ultraviolet light emitted by the ultraviolet light source is 315-400nm.

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

[0015] The invention provides an application of psoralen in preparing a medicine for treating vitiligo. The invention adopts the flavonoid compound psoralen to prepare the medicine for treating vitiligo, which has low toxicity and has a good therapeutic effect on vitiligo in combination with ultraviolet light (such as UVA). BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a comparison chart of liquid chromatography of psoralen and adenine before and after illumination;

[0017] Figure 2 This is a comparison chart of liquid chromatography of psoralen and guanine before and after illumination;

[0018] Figure 3 This is a comparison chart of liquid chromatography of psoralen and thymine before and after illumination;

[0019] Figure 4 This is a comparison chart of liquid chromatography of psoralen and cytosine before and after illumination;

[0020] Figure 5 This is the liquid chromatography diagram of the photo-action of psoralen A on thymine / AT-DNA;

[0021] Figure 6 This is the result of analyzing the photoaddition of psoralen to thymine by LC-MS;

[0022] Figure 7 The figure is the inferred structure of the photoadduct and its fragmentation law;

[0023] Figure 8 This is a diagram showing the color change of the solution of psoralen and AT-DNA after being exposed to light for different time periods;

[0024] Fig. 9 The UV absorption spectra of psoralen and thymine at different illumination times under the condition of thymine excess;

[0025] Fig.10 The UV absorption spectra of psoralen and thymine at different illumination times under the condition of psoralen excess;

[0026] Fig.11 The UV absorption spectrum of AT-DNA was gradually added to a certain concentration of psoralen;

[0027] Fig.12 The figure is a comparison of the UV absorption spectra of psoralen, AT-DNA and its photoadduct;

[0028] Fig.13 This is the infrared absorption spectrum of psoralen A acting on thymine;

[0029] Fig.14 is the femtosecond transient absorption spectrum of psoralen after excitation at 340 nm;

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

[0031] Fig.16 One-dimensional and two-dimensional conductivity signal diagrams of BA;

[0032] Fig.17 The one-dimensional and two-dimensional conductivity signal diagrams of AT-DNA;

[0033] Fig.18 The one-dimensional and two-dimensional conductivity signal diagrams of ATUV;

[0034] Fig.19 One-dimensional and two-dimensional conductivity signal diagrams of BAAT;

[0035] Fig. 20 The one-dimensional and two-dimensional conductivity signal diagrams of BAATUV;

[0036] Fig.21 is the molecular length of the pull-up compound;

[0037] Fig. 22 This is a test result of the therapeutic effect of psoralen A on the progression of vitiligo;

[0038] Fig.23 This is a graph showing the anti-inflammatory ability test results of psoralen;

[0039] Fig.24 This is the result diagram of the effect of psoralen on oxidative stress and CD8 T cell infiltration in mice;

[0040] Fig.25 The toxicity test results of psoralen A in treating vitiligo;

[0041] Fig.26 The schematic diagram is a mechanism of action of psoralen A in treating vitiligo according to the present invention. DETAILED DESCRIPTION

[0042] The invention provides an application of psoralen in preparing a medicine for treating vitiligo.

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

[0044]

[0045] At present, in the related art, PUVA therapy is used to treat vitiligo, and coumarin compounds or their derivatives are usually used as active agents, such as psoralen (CAS No. 66-97-7, structural formula shown in Formula A), 8-methoxypsoralen (CAS No. 298-81-7, structural formula shown in Formula B), 5-methoxypsoralen (CAS No. 484-20-8, structural formula shown in Formula C) or 4,5',8-trimethylpsoralen (CAS No. 3902-71-4, structural formula shown in Formula D). However, the use of the above coumarin compounds as active agents is highly toxic and may cause some adverse reactions, such as nausea, vomiting, stomach discomfort, headache, insomnia, nervousness, nausea, nausea, depression, etc. At present, there are few studies on flavonoid compounds, and there is no relevant research on the effect of psoralen A in preventing and treating vitiligo.

[0046]

[0047] The present invention adopts the flavonoid compound psoralen to prepare a medicine for treating vitiligo, which has low toxicity and has a good therapeutic effect on vitiligo when combined with ultraviolet light (such as UVA). Specifically, in the embodiment of the present invention, liquid chromatography-mass spectrometry (LC-MS) is used to predict the possible binding mode of psoralen with DNA, and then a multispectral method is used, including ultraviolet-visible (UV-vis) absorption spectroscopy, infrared (IR) absorption spectroscopy and femtosecond transient absorption spectroscopy (fs-TA) and ultra-high performance liquid chromatography (UPLC) and STM-BJ to determine the interaction between psoralen and DNA, wherein the use of liquid chromatography-mass spectrometry and ultra-high performance liquid chromatography analysis can provide the molecular weight and rich structural information of each component, making the qualitative analysis results more reliable and the analysis time fast; femtosecond transient absorption spectroscopy can perform time-resolved data analysis, which is suitable for complex photophysical and photochemical processes; STM-BJ technology can realize the accurate measurement of the conductivity of a single molecule, which is crucial for understanding the charge transfer mechanism when molecules interact with DNA. This technology can quickly construct thousands of single-molecule junctions in a short period of time, capture the dynamic change process of a single molecule, and present it in the form of an electrical signal, with the characteristics of good repeatability and high time accuracy. At the same time, based on the determination that psoralen can bind to thymine bases, the present invention studies the therapeutic effect of psoralen on vitiligo, reveals the potential mechanism of psoralen in treating vitiligo, and proposes guidelines for the reasonable improvement of PUVA active agents. Moreover, the present invention uses psoralen as an active agent, and its hepatotoxicity and nephrotoxicity are less than the toxicity of coumarin compounds currently used in clinical practice.

[0048] In an embodiment of the present invention, the drug may include psoralen and a pharmaceutically acceptable carrier; the present invention has no special limitation on the specific type of the pharmaceutically acceptable carrier, and any type of pharmaceutically acceptable carrier familiar to those skilled in the art may be used; the content of psoralen in the drug may be 0.1 to 10 wt%, further 0.5 to 5 wt%, further 0.8 to 2 wt%, and further 0.9 to 1.5 wt%.

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

[0050] In the examples of the present invention, vitiligo induced by Monobenzone cream was specifically used to verify the efficacy of psoralen A in the present invention on vitiligo.

[0051] The present invention provides a system for treating vitiligo, comprising an ultraviolet light source and a chemical drug, wherein the chemical drug is a drug comprising psoralen A. In an embodiment of the present invention, the chemical drug may be the drug for treating vitiligo as described in the above technical solution, which will not be described in detail herein.

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

[0053] The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0054] Some of the materials used in the following examples are as follows:

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

[0056] All data involved in the statistical analysis in the following examples are expressed as mean ± standard error of at least three independent experiments. One-way ANOVA was used for multiple group comparisons, and Student's t test (two-tailed) was used for two-group comparisons. Statistical analysis was performed using GraphPad Prism software. P values ​​< 0.05 indicated statistical significance.

[0057] Example 1 UPLC analysis

[0058] Analytical methods:

[0059] The analysis was performed on a Waters ACQUITY UPLC system. The system was controlled by Empower Pro software (Waters). An XBridge Premier Oligonucleotide BEH C18 column (4.6×150 mm, 2.5 μm) was used for gradient elution at 30°C. The mobile phase included mobile phase B and mobile phase D, wherein mobile phase B was acetonitrile and mobile phase D was a 0.1% volume fraction of formic acid in water. The gradient elution program was as follows: from 0 to 10.67 min, the volume fraction of mobile phase B increased from 5% to 50% at a uniform rate; from 10.67 to 19.67 min, the volume fraction of mobile phase B increased from 50% to 95% at a uniform rate; from 19.67 to 23.42 min, the volume fraction of mobile phase B decreased from 95% to 5% at a uniform rate. The detection wavelength used 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 example, the photoaddition of psoralen to 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 chart of liquid chromatography of psoralen and adenine before and after illumination. Figure 2 This is a comparison chart of the liquid chromatography of psoralen and guanine before and after illumination. Figure 3 This is a comparison chart of liquid chromatography of psoralen and thymine before and after illumination. Figure 4 The figure is a comparison of the liquid chromatography of psoralen and cytosine before and after illumination, where orange represents the liquid chromatography before illumination and blue represents the liquid chromatography after illumination. The results show that no new compound peaks are generated in the mixed solution of psoralen and A and G bases before and after illumination (such as Figure 1 and Figure 2 As shown); the mixed solution of psoralen and T and C bases has a new peak after a certain period of light exposure, and this peak is located between the peaks of psoralen and thymine. It is speculated that the new peak is due to the generation of photoadducts (as shown Figure 3 and Figure 4 However, the peak area of ​​the photoadduct bound to thymine was larger than that bound to cytosine, indicating that psoralen can bind to DNA and preferentially bind to thymine to form a photoadduct.

[0062] On the basis of confirming that it can bind to thymine, this example then analyzes the liquid chromatograms of psoralen with thymine and AT-DNA at different illumination times. Figure 5 This is the liquid chromatography diagram of the photo-action of psoralen A on thymine / AT-DNA; Figure 5 A in the figure is the liquid chromatogram of psoralen and thymine at different illumination times. Figure 5 B in the middle is the liquid chromatogram of psoralen A at different illumination times. Figure 5 C in the middle is the liquid chromatogram of psoralen and AT-DNA at different illumination times. Figure 5 D is a comparison of the liquid chromatography of psoralen A with different ratios of thymine and psoralen A after 96 hours of illumination, where peaks 1 and 2 are thymine, peak 4 is psoralen A, and peak 3 is the photoadduct. The results show that with the increase of illumination time, peak 4 gradually decreases, peak 3 gradually increases and reaches the maximum at 96 hours of illumination (e.g. Figure 5 Replace thymine with AT-DNA (as shown in A). Figure 5 The results shown in C) and Figure 5 The liquid chromatograms of psoralen A at different illumination times were used as controls (e.g. Figure 5 In order to increase the yield of photoadduct formation, different ratios of psoralen to thymine were screened to form photoadducts (such as Figure 5 The results show that after 96 hours of illumination, when the mass ratio of psoralen to thymine is 1:10 or 1:20, the peak area of ​​peak 3 is larger, that is, the yield of photoadduct formation is higher.

[0063] Example 2 LC-MS analysis

[0064] Analytical methods:

[0065] Samples were analyzed on 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 in positive and negative modes using electrospray ionization with the following source conditions: ion source was HESI, sheath gas rate was 40 arb, auxiliary gas rate was 10 arb, spray voltage was 3.8 kV for positive ions and 3.0 kV for negative ions, capillary temperature was 320 °C, auxiliary gas temperature was 300 °C, scan mode was Full ms / dd-ms2top10, and collision voltage was NCE15, 30, 45. Chromatographic separation is achieved by injecting 5 μL of sample at a flow rate of 0.5 mL / min on an Eclipse Plus C18 chromatographic column (100 mm×4.6 mm, 3.5 μm), wherein the mobile phase comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is a formic acid aqueous solution with a volume fraction of 0.1%, and the mobile phase B is acetonitrile; a gradient elution is adopted, and the gradient elution program is as follows: from 0 to 13 min, the volume fraction of the mobile phase B is increased uniformly from 5% to 50%; from 13 to 25 min, the volume fraction of the mobile phase B is increased uniformly from 50% to 95%; and from 25.1 to 30 min, the volume fraction of the mobile phase B is decreased uniformly from 95% to 5%.

[0066] Results and Discussion:

[0067] In this example, the photobinding of psoralen to thymine was further analyzed by LC-MS. Figure 6 The results of the photoaddition of psoralen to thymine by LC-MS analysis are shown in Figure 1, where A is the total ion current of negative ions of psoralen and thymine before and after illumination, B is the total ion current of positive ions of psoralen and thymine before and after illumination, C is the compound cleavage fragment information of psoralen, D is the compound cleavage fragment information of thymine, E is the secondary mass spectrometry retention time diagram of the photoadduct, and F is the cleavage fragment information diagram of the photoadduct. The results show that after 96 hours of illumination, the absorption intensity of psoralen decreases, and new peaks are generated (such as Figure 6 A and B in the figure). Figure 6 In the figure, C and D are the pyrolysis fragment information of psoralen and thymine, respectively. By searching the relative molecular mass of thymine, 127.0506, in the secondary mass spectrum, the result shows that there is a compound with a relative molecular mass of 451.1874 at 12.65min. Its pyrolysis fragment information contains the pyrolysis fragment information of psoralen and thymine (such as Figure 6 (As shown in E and F), this indicates that psoralen can react with thymine to produce photoadducts under light excitation.

[0068] Figure 7 Figure 3 is the inferred structure of the photoadduct and its fragmentation pattern. The results show that under the excitation of light, the isopentenyl double bond of psoralen and the carbon-carbon double bonds at positions 5 and 6 of thymine base break and then combine to form a cyclobutane pyrimidine dimer.

[0069] Example 3 Spectral characteristics

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

[0071] Analytical methods:

[0072] UV-Vis absorption spectra were obtained using a UV-2600. Solutions were measured in fused quartz cuvettes from Hellma (path length 1 cm). In UV / Vis experiments, the effect of light addition on the UV absorption spectrum was investigated by irradiating the sample solution at 365 nm with a xenon lamp. The solutions were irradiated in a beaker under magnetic stirring conditions. 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. IR spectra were measured using a Bruker Vertex 70V IR spectrometer. The IR spectra were obtained using a CaF 2 The measurements were performed at room temperature (20-21°C) using a custom-made cuvette with a window (path length of 100 μm).

[0073] Results and Discussion:

[0074] 1.1. UV-Vis absorption characteristics of photoaddition

[0075] UV spectroscopy is one of the most commonly used methods to detect the interaction between small molecules and DNA. Figure 8 The figure is a color change diagram of the solution of psoralen and AT-DNA at different illumination times. The results show that after psoralen and thymine are mixed, the color of the solution gradually deepens with the increase of illumination time until it becomes dark yellow.

[0076] Fig. 9 The UV absorption spectra of psoralen and thymine at different illumination times under the condition of excess thymine are shown in the figure. The results show that in the case of excess thymine, the UV absorption is mainly dominated by the absorption of thymine, and thymine exhibits two absorption bands near 215nm and 263nm. As the illumination time increases, the absorption intensity of thymine at 215nm and 263nm decreases and shows blue shift and red shift respectively, and the absorption intensity between 350 and 425nm gradually increases, which is not seen before illumination.

[0077] Fig.10The UV absorption spectra of psoralen and thymine at different illumination times under the condition of excess psoralen are shown in the figure. The results show that under the condition of excess psoralen, the UV absorption is mainly dominated by the absorption of psoralen, and psoralen exhibits three absorption bands near 220nm, 277nm and 325nm. With the increase of illumination time, the absorption of psoralen at 220nm, 277nm and 325nm becomes flat, which is also absent before illumination.

[0078] Fig.11 The UV absorption spectrum of AT-DNA is gradually added to a certain concentration of psoralen. In order to further verify the photoaddition reaction between the compounds, the present embodiment studies the UV absorption spectrum of psoralen in the absence and presence of different concentrations of DNA. The results show that as the DNA content increases, the absorption intensity of psoralen at 220nm, 277nm and 325nm decreases and the absorption intensity between 350-425nm increases.

[0079] Fig.12 This is a comparison chart of the ultraviolet absorption spectra of psoralen, AT-DNA and photoadduct. The results show that the ultraviolet absorption spectra of the three are obviously different.

[0080] The above results strongly prove that psoralen can interact with DNA and form photoadducts (i.e., BA-DNA complexes).

[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. Fig.13 This is the infrared absorption spectrum of psoralen A acting on thymine; Fig.13 A in the figure is the IR spectrum of psoralen. Fig.13 Where B is the IR spectrum of thymine, Fig.13 C in the figure is the IR spectra of psoralen and thymine at different illumination times. Fig.13 D in the figure is the IR comparison chart of psoralen, thymine and photoadduct. Fig.13 As shown in A and B, the spectrum of psoralen is at 1714 cm -1 There is a vibration band at 1651cm, which is attributed to the carbonyl stretching vibration; -1 A wide band of 1579cm -1 A broad band at 1731 cm is attributed to C=C stretching vibration. Thymine has three distinct bands in the high frequency range. -1 and 1674cm -1The band at 1600 cm-1 can be assigned to the carbonyl stretching vibration. -1 The band at can be assigned to C=C stretching vibration. In the buffer of psoralen and thymine, about 95% of psoralen is inserted. The initial spectrum of BA-T before illumination is dominated by the vibration of thymine. Fig.13 As shown in C and D, when irradiated with a xenon light source system emitting at 365 nm, it causes a change in the infrared spectrum. -1 The absorption changes around 1640 cm-1 may be due to the carbonyl stretching vibration of psoralen and the bleaching of thymine. -1 The absorption change around 500cm is probably due to the vibration of thymine. -1 The absorption intensity of the nearby band increases with the extension of illumination time and shifts slightly to higher wavenumbers. This temporal behavior indicates that it is due to secondary photochemistry, namely the formation of cross-links.

[0083] 2. Femtosecond transient absorption spectroscopy

[0084] Analytical methods:

[0085] Femtosecond transient absorption (fs-TA) data were acquired by the femtosecond laser pharos transient absorption spectroscopy harpia, and the laser system Pharos provided the basic light source. The absorbance change of the pump was used for detection, and the detection beam was set to the magic angle. The time resolution was 180fs. The excitation wavelength was 340nm and the pump power was 1μJ / cm 2 , with a detection range of 370 to 500 nm, a repetition rate of 5 Hz and a pulse duration of 8 ns for photoexcitation. The instrumental time zero offset was determined by the optical Kerr effect as a function of wavelength and corrected. The signal of the pure solvent was recorded in a separate measurement and subtracted after appropriate scaling to eliminate time zero artifacts. The solution was circulated through a fused silica flow cell with a path length of 1 mm. The sample volume was large enough to make the contribution of the photoproducts formed during the experiment negligible.

[0086] Results and Discussion:

[0087] The BA solution containing AT-DNA was excited with a UVA laser pulse and detected in the UV / Vis region. The BA and AT-DNA solution was placed in a buffer and excited with a 340 nm laser pulse. For the excitation wavelength used, no absorption below 340 nm could be obtained. Fig.14 This is the femtosecond transient absorption spectrum of psoralen after excitation at 340 nm. Fig.15The femtosecond transient absorption spectrum of BA-DNA adduct after excitation at 340nm. The results show that near time zero, the BA solution has an obvious positive absorption at 390-470nm and reaches a peak at 430nm, and an obvious negative absorption at 373nm (such as Fig.14 Adding AT-DNA to the BA solution will change the decay kinetics of BA (as shown in Fig.15 As shown in Figure 2, the positive absorption at 390-470nm and the negative absorption at 373nm were significantly reduced, and the positive absorption below 390nm and above 480nm increased. The kinetic curve and SPE spectrum also showed the same result. The above results indicate the formation of photoadducts.

[0088] 3. Conductivity measurement

[0089] Analytical methods:

[0090] The conductivity of the molecular junction was measured using a Scanning Tunneling Microscope-Break Junction (STM-BJ) instrument. The BAATUV compound was produced by repeatedly forming and disconnecting the atomic contact between a gold tip and a gold substrate with a purity of 99.99%. At room temperature, repeated opening and disconnection cycles were performed in a mixed solution of psoralen A and DNA containing 1 μM of the target molecule, and a bias of 100 mV was applied between the gold tip and the gold substrate. The frequency of the current-voltage converter used for data acquisition was 50 kHz. During all the break junction measurements in this embodiment, the sample was immersed in a solvent, and the gold tip and the gold substrate were still immersed in the solvent at the end of all measurements.

[0091] Results and Discussion:

[0092] For conductance measurements, the target molecule solution was dropped on the gold substrate, while the gold STM-BJ probe suspended on top of the gold substrate was approached to the substrate by a stepper motor control and a piezoelectric stack to form an Au-Au point contact. The molecules bound the gold tip and substrate to form a stacked supramolecular junction. All molecules were dissolved in PBS buffer at a concentration of 1 μM. Conductance measurements were performed at room temperature with a bias of 0.1 V applied between the gold tip and the substrate. The flow chamber was illuminated with a UV light source with a wavelength of 365 nm, and the conductance traces obtained during the break junction measurement of each molecule were collected to construct one-dimensional and two-dimensional conductance signal maps.

[0093] Fig.16 is the one-dimensional and two-dimensional conductivity signal diagram of BA, Fig.17 The one-dimensional and two-dimensional conductivity signal diagrams of AT-DNA are shown in Figure 1. Fig.18 The one-dimensional and two-dimensional conductivity signal diagrams of ATUV, Fig.19The one-dimensional and two-dimensional conductivity signal diagrams of BAAT, Fig. 20 The one-dimensional and two-dimensional conductivity signal diagrams of BAATUV, Fig.21 is the molecular length of the pull-up compound. The comparison between two groups was performed by t-test, and multiple groups were compared by one-way analysis of variance; *p<0.05, **p<0.01, ***p<0.001. The results showed that the one-dimensional and two-dimensional conductivity signal intensities of BA, AT, ATUV, and BAAT were 10 -4 ~10 -2 Between (such as Figure 16 to Figure 19 The conductivity signal of the mixed solution of BA and AT-DNA was significantly enhanced under light excitation (as shown in Fig. 20 The increase in the conductivity signal indicates that under the excitation of light, BA binds to AT-DNA to produce photoadducts. Then the length of the stretched molecules in each group was statistically analyzed, as shown in Figure 2. Fig.21 As shown in the figure, the molecular length of BA and AT-DNA increased significantly after illumination. The significant extension of the molecular length indicates that the binding of BA increases the length of AT-DNA by inserting into AT-DNA.

[0094] Example 4 Animal Experiment

[0095] Analytical methods:

[0096] 1. Model establishment and drug administration scheme of vitiligo mice

[0097] Six-week-old male C57BL / 6 mice were purchased from Henan Sikebes Biotechnology Co., Ltd. (SCXK (Yu) 2020-0005). Animal ethics were approved by the Ethics Committee of the First Affiliated Hospital of Shihezi University. The animals were kept in a temperature-controlled environment, the temperature was maintained in the range of 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 adaptively fed for 7 days before the start of the experiment. Before the start of the experiment, a total of 70 C57BL / 6 mice were shaved on the back with an electric razor, covering 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. The control group applied an equal amount of sterile water to the depilated area. An experimental vitiligo mouse model was established for 20 consecutive days. After modeling, the mice were divided into model group, 8-MOP group, Et-OH group, UV group, BA group, and BAUV (i.e., psoralen + light) group, with 10 mice in each group. The drug was applied once a day for 28 consecutive days starting from the 21st day of modeling, 1 mL each time. Subsequently, the skin lesions were scored using the bleaching scoring method, and skin tissue and serum were collected on the last day of treatment for further analysis. The vitiligo model was evaluated using a scoring system based on the degree of depigmentation in the treatment area. 1 point means that the depigmentation area is less than 10%, 2 points means that the depigmentation area is less than 10-25%, 3 points means that the depigmentation area is less than 25-50%, 4 points means that the depigmentation area is less than 50-75%, and 5 points means that the depigmentation area exceeds 75%. White hair counts were performed by visually inspecting the dorsal area of ​​each mouse and counting the number of white hair clusters present using Image J. These methods were used to evaluate the effectiveness of vitiligo induction and quantify the degree of depigmentation and white hair appearance in experimental mice.

[0098] 2. Histological analysis

[0099] The skin tissue of the depigmented area was fixed in 4wt% paraformaldehyde buffer, dehydrated with a series of ethanol (75%, 85%, 95% and 100%) and xylene, and embedded in paraffin. The treated tissue was then cut into 5μm sections. HE, Masson and Masson-Fontana staining were performed on these sections to accurately determine the melanin content. These staining schemes laid a reliable foundation for further exploration of tissue structure and were conducive to the preliminary evaluation of melanin content and related structures.

[0100] 3. Evaluation of Antioxidant Stress and Anti-Inflammatory Activities in Skin Tissues of Vitiligo Mice

[0101] After BAVACHIN treatment, mouse skin tissue was collected, tissue homogenate was obtained using the extraction solution provided with the kit, and the supernatant was collected. Subsequently, the activity of superoxide dismutase (SOD) and catalase (CAT), as well as the concentration of malondialdehyde (MDA) were quantified in this experiment, and the level of antioxidant stress was evaluated using a commercial detection kit. CAT and SOD activities were measured using a SOD activity detection kit (JL-T0781, Jianglai Biology) and a CAT activity detection kit (JL-T0900, Jianglai Biology), respectively. MDA concentration was measured using an MDA content determination kit (JL-T0761, Jianglai Biology) according to the protocol provided by the manufacturer. At the same time, mouse blood was collected by centrifugation (4°C, 3000rpm, 15min), and then serum was collected. Then, commercial ELISA detection kits purchased from Uni-President 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 according to the recommended procedures provided by the manufacturer to evaluate its anti-inflammatory activity.

[0102] 4. Immunofluorescence staining

[0103] For immunofluorescence analysis of skin tissue, a small piece of skin was removed from the depigmented area on the back of the mouse. The skin tissue was fixed in 4wt% paraformaldehyde for 24h at 4°C and then dehydrated in 30wt% sucrose solution at 4°C. Subsequently, the tissue was frozen in optimal cutting temperature (OCT) compound and cut into 15μm sections using a cryostat (RWD FS800A, Shenzhen, China). The sections were washed three times with PBS to remove OCT and then immunostained with AlexaFluor 647CD8a (100724, BioLegend, San Diego, CA, USA, 1:200) to detect CD8 T cells in 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 GoatAntiRabbit IgG (H&L)-AlexaFluor 594 (RS3611, Immunoway, Plano, TX, USA, 1:200) to assess the levels of inflammatory factors in skin tissue. All sections were imaged using a laser scanning confocal microscope. Finally, the acquired data were analyzed using Image J 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 example used a vitiligo mouse model induced by 4% monobenzone cream to investigate the potential of BA to intervene in the progression of vitiligo. Fig. 22 The results of the test of the therapeutic effect of psoralen on the progression of vitiligo are shown in Figure 1, where A is a schematic diagram of the animal model and treatment; B is a result of monitoring the depigmentation of the back of mice, and the white arrow indicates the white hair area; C is a depigmentation score of vitiligo mice, and D is a statistical diagram of the number of white hairs after treatment, and the data are expressed as standard error ± mean (n = 6); E is a result of HE staining, Masson staining, and Masson-Fontana staining of mouse skin tissue, and representative images are shown in the figure, with a scale of 200 μm. The t-test was used for comparison between the two groups, and one-way analysis of variance was used for multiple groups; *p<0.05, **p<0.01, ***p<0.001.

[0107] The results showed that after the vitiligo model was established (day 0), mice in each drug-treated group were treated and then killed after four weeks of treatment (e.g. Fig. 22 The extent of hair depigmentation on the back of mice was assessed by visual observation. The hair on the back of mice showed significant pigment loss after application of monobenzone cream to the designated areas compared with the normal group. However, after intervention, a decrease in pigment loss was observed in the BAUV group, which was further confirmed by statistical analysis (see Figure 2A). Fig. 22 Statistical analysis of the depigmentation scores after modeling mice showed that the vitiligo group had a significant increase in depigmentation scores, while those treated with BAUV (vitiligo + BAUV) showed a significant decrease in scores, highlighting the significant difference between the two groups (see Figure 2A). Fig. 22 In addition, the number of white hairs in the normal group, vitiligo group and each treatment group was calculated in this example. The results showed that the number of white hairs on the back of mice in the vitiligo+BAUV group was significantly less than that in the vitiligo group (as shown in Figure 2A). Fig. 22 (as shown in D). In order to evaluate the histological changes in skin tissue, Masson-Fontana staining and HE staining were performed in this example to examine the dorsal skin of vitiligo mice, especially the changes in melanin granules. HE staining results showed that the skin of mice in the vitiligo group 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 decrease in pathological changes. In Masson-Fontana staining, melanin undergoes a silver reaction, resulting in the formation of black granules. After four weeks of intervention, the amount of melanin around the hair follicles in the treatment group increased significantly. In addition, the number of melanin granules that migrated to the skin surface increased significantly (as shown in Figure 5A). Fig. 22Together, these findings 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 treated groups.

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

[0109] The level of inflammatory factors is a key indicator for detecting vitiligo patients. Therefore, measuring the level of inflammatory factors is a common method for evaluating the progress and effect of vitiligo treatment. Fig.23 The results of the anti-inflammatory ability test of psoralen are shown in Figure 1, where A, B and C are the concentrations of TNF-α, IFN-γ and IL-6 in the serum of mice after 28 days of psoralen treatment, respectively. D and E are immunofluorescence images of inflammatory factors in the skin of mice in different groups (blue: DAPI; red: TNF-α, IFN-γ), and the scale bar is 100μm. The comparison between the two groups was performed by t-test, and the comparison between multiple groups was performed by one-way analysis of variance; *p<0.05, **p<0.01, ***p<0.001.

[0110] The results showed that proinflammatory cytokines (such as TNF-α, IFN-γ, and IL-6) were significantly increased in the serum of mice with monobenzone-induced vitiligo. However, after BAUV treatment, the levels of these inflammatory factors were significantly reduced (such as Fig.23 In addition, immunofluorescence staining was performed on the skin of the depigmented area of ​​mice in this example, and 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 (such as Fig.23 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] Fig.24 The results of the effect of psoralen on oxidative stress and CD8 T cell infiltration in mice are shown in Figure 1, where A, B and C are the CAT activity test, SOD activity test and MDA content test in mouse skin tissue after 28 days of psoralen treatment, respectively; D is the immunofluorescence image of skin CD8 T cells after 28 days of psoralen treatment (blue: DAPI; red: CD8+T cell), with a scale of 50 μm. The t-test was used for comparison between the two groups, and one-way analysis of variance was used for multiple groups; *p<0.05, **p<0.01, ***p<0.001.

[0113] This example evaluates the concentration of malondialdehyde (MDA) and the activities of superoxide dismutase (SOD) and catalase (CAT) in mouse skin tissue after intervention. Fig.24As shown in A, B, and C, oxidative stress in the skin caused a sharp drop in SOD and CAT levels and a significant increase in MDA levels in the vitiligo group. However, after BAUV treatment, the MDA concentration was significantly reduced, and the SOD and CAT activities were restored (p<0.05). During the occurrence of vitiligo, CD8 T cells are enriched in the dermis and cause the destruction of melanocytes, which may affect the production of melanin and is one of the possible factors in the pathogenesis of vitiligo. In order to explore the relationship between BAUV and immune infiltration, this example conducted an in-depth study on CD8 T cell infiltration, specifically, immunofluorescence staining was performed on the skin in the depigmented area to evaluate the infiltration of CD8 T cells. As shown in Fig.24 As shown in D, the number of CD8 T cells in the skin of the vitiligo group was greater than that in the skin of the other two groups.

[0114] 4. Toxic effects of BAUV on vitiligo mice

[0115] Fig.25 The toxicity test results of psoralen A in the treatment of vitiligo, where A is a photo of mouse liver, spleen, and kidney, B, C, and D are organ coefficient results of mouse liver, kidney, and spleen, respectively, and E is HE staining results of mouse liver, spleen, and kidney. The two groups were compared using t-test, and multiple groups were compared using one-way analysis of variance; *p<0.05, **p<0.01, ***p<0.001.

[0116] In this example, the organs of vitiligo mice after BAUV treatment were visually observed and evaluated. Fig.25 As shown in Figure A, compared with the blank control group, the livers of the 8-MOP group, BA group, and BAUV group were damaged to a certain extent, and there were no significant differences in the spleen and kidney of each group. Fig.25 As shown in Figures B, C and D, compared with the Control group, the liver weights of the 8-MOP group, the BA group and the BAUV group increased significantly, while the kidney weights of the 8-MOP group and the BA group also increased to a certain extent. The spleen weights of the drug-treated groups were not statistically significant. In order to evaluate the histological changes of various organ tissues, HE staining was performed in this example to examine the organ changes of vitiligo mice. Fig.25As shown in E, the liver tissue structure of the Control group was basically normal, the hepatocytes were normal in morphology, the nuclei were clear, the cytoplasm was abundant, and there was no edema or fatty degeneration; the hepatocytes were arranged tightly and regularly, no obvious inflammatory cell infiltration was observed, and the hepatic sinusoids were not dilated. The liver tissue structure of the 8-MOP group was severely abnormal, with a large number of inflammatory cells infiltrating in the hepatic sinusoids, mild edema in some hepatocytes, and vacuolated cytoplasm. The liver tissue of the BA group was moderately abnormal, with mild dilation of some hepatic sinusoids and vacuolated cytoplasm. The liver tissue structure of the BAUV group was mildly abnormal, with tight and regular arrangement of hepatocytes, no obvious inflammatory cell infiltration, and vacuolated cytoplasm in some parts. The glomeruli and tubules of the Control group were normal in structure, with no proliferation of mesangial cells and matrix, and no inflammatory cell infiltration and fibrosis in the renal interstitium; some glomeruli in the 8-MOP group and the BA group were significantly enlarged or reduced, and there was no significant difference in renal tissue between the BAUV group and the Control group. There was no significant difference in the spleen of each group except the Model group. In the Model group, the junction between the red pulp and the white pulp was blurred, and there was no hyperplasia of the splenic capsule and trabecular fibrous connective tissue, and there was no inflammatory cell infiltration.

[0117] Fig.26 The present invention is a schematic diagram of the mechanism of action of psoralen A in treating vitiligo, specifically, psoralen A can effectively treat vitiligo while binding to DNA thymine bases, and the mechanism of this treatment involves regulating oxidative stress, restoring antioxidant capacity, and inhibiting inflammatory response. Psoralen A treatment effectively reduces depigmentation, promotes the formation of melanin particles, and contributes to good histological changes in the skin of vitiligo mice.

[0118] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An application of psoralen in the preparation of a medicine for treating vitiligo.

2. The use according to claim 1, characterized in that: The medicine comprises psoralen and a pharmaceutically acceptable carrier.

3. The use according to claim 2, characterized in that: The content of psoralen in the medicine is 0.1-10wt%.

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

5. The use according to claim 4, characterized in that: The administration method of the drug includes administration by application.

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

7. A system for treating vitiligo, comprising an ultraviolet light source and chemical drugs, wherein the chemical drugs are drugs including psoralen.

8. The system according to claim 7, characterized in that The wavelength of the ultraviolet light emitted by the ultraviolet light source is 315-400nm.

9. The system according to claim 8, characterized in that The wavelength of the ultraviolet light is 365 nm.

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