CN113768921A

CN113768921A - Application of methoxy flavonoid compound in preparing anti-leucoderma medicament

Info

Publication number: CN113768921A
Application number: CN202111090110.8A
Authority: CN
Inventors: 张波; 黄思露; 王晓琴
Original assignee: Individual
Current assignee: Individual
Priority date: 2021-09-11
Filing date: 2021-09-11
Publication date: 2021-12-10

Abstract

The methoxy flavonoid compound with 2-phenyl chromone mother nucleus has the function of resisting leucoderma, and represents compounds of kaempferide, isorhamnetin and kaempferol 3, 4' -dimethyl ether. The methoxy flavone compound promotes the regeneration and the new generation of melanocytes by promoting the expression of key genes in a melanin generation path, induces the accumulation of melanin, maintains the melanin homeostasis by up-regulating the expression of melanin homeostasis-related protein adcy6, and reduces the decomposition of the pigment of the original melanocytes; meanwhile, the increase of active oxygen under physiological dose is induced, the expression of related genes such as gclm, gclc and the like synthesized by glutathione is reduced, and the melanin metabolism is reduced, so that the function of treating leucoderma is exerted, and the methoxy flavonoid compound is suggested to have important value and development prospect in the research and development of leucoderma treatment drugs.

Description

Application of methoxy flavonoid compound in preparing anti-leucoderma medicament

Technical Field

The invention relates to a new application of methoxy flavonoid compounds, namely the application in the aspect of preparing anti-vitiligo drugs.

Background

Vitiligo is acquired pigment disorder with unknown reasons, no gender and ethnicity difference, and no age difference of the ill people. The clinical characteristics are that the patient forms white or pale plaques with clear hypopigmentation or disappearance in the acquired boundary, the skin color at the edge of the skin lesion and the skin color near the edge of the skin lesion is usually unchanged or slightly deepened, and the hair at the diseased part is often changed into white due to the hypopigmentation. According to the European consensus, it can be divided into two main classes, Segmental (SV) and non-segmental (NSV). Vitiligo is one of three persistent skin diseases in the world, the global morbidity of the vitiligo is about 0.5% -2%, the morbidity of partial areas can reach 2% -3%, and the morbidity of China is about 0.1% -2%. Although leucoderma is not a fatal disease, the white spot exposed on the body surface often seriously affects the beauty of the human body, and often brings psychological disorders such as social fear, self-assessment decline, anxiety and even depression to patients, meanwhile, the leucoderma may also be complicated with other diseases such as hyperthyroidism, alopecia areata, pernicious anemia and the like, and the quality of life of the patients is seriously affected.

The pathogenesis of vitiligo is not clear at present, and is closely related to various factors such as heredity, immunity, oxidative stress, melanocyte destruction and the like, but no theory can explain the occurrence of the disease. In recent years, most studies indicate that the pathogenesis of vitiligo is not univocal, and the hypothesis with the highest support rate to date is the autoimmune hypothesis after the theory of oxidative stress. Reactive oxygen species accumulation in the skin directly destroys melanocytes, leading to apoptosis. The reactive oxygen species may cause the transfer of calreticulin from the lumen of the endoplasmic reticulum to the cell surface, the secretion of HSP70i and the expression of IL-6, IL-8 and TNF- α in stressed melanocytes. Langerhans Cells (LCs) which are tumor necrosis factor-related apoptosis-inducing ligand receptors on melanocytes also cause melanocytes to apoptosis. According to the present studies, vitiligo is mainly manifested as depigmentation of the skin, caused by a reduction or loss of functional melanocytes of the skin and/or hair follicles.

The final purpose of western medicine treatment of vitiligo is to promote melanin cells in the skin epidermis to secrete melanin and migrate to the damaged stratum corneum, thereby protecting the stratum corneum cells. The current treatment methods are mainly divided into non-operative treatment (including drug treatment and physical treatment) and surgical treatment or combination of a plurality of treatment modes.

The drug treatment of vitiligo is mainly divided into local drug administration and integral drug administration, wherein the drug treatment mainly comprises glucocorticoid, immunosuppressant, photosensitizer and the like, the effective rate is 60%, the cure rate is 7%, and the incidence rate of adverse reactions is 69%. Glucocorticoids have the disadvantage of having large side effects, immunosuppressants cause local adverse reactions such as skin burning or itching, the expected effects of drugs for treating depigmentation are difficult to ensure, the depigmentation time is long, and new white spots are induced, while the short-term use of photosensitizers has side effects of causing erythema and skin burning, and the long-term use of photosensitizers has side effects including skin malignancy change.

Physical therapy mainly includes ultraviolet therapy, 308nm excimer laser, helium neon laser, and photochemotherapy. Narrow-spectrum medium-wave ultraviolet therapy (NB-UVB) is highly phototoxic, and induces melanin accumulation and melanocyte death. 308nm excimer laser, helium neon laser and photochemotherapy have great clinical popularization difficulty, and the treatment method has no universality.

The surgical treatment comprises surgical excision and autologous skin transplantation, the surgical excision has two modes of skin expansion and skin flap transfer, and is suitable for patients with vitiligo with small skin damage area, and patients with relatively large area are not suitable for surgical excision treatment. Autologous skin grafts can be divided into three categories: normal skin (epidermis or epidermis and dermis) transplantation containing melanocytes, non-cultured epidermis or hair follicle transplantation containing melanocytes, and transplantation of melanocytes (with or without keratinocytes) grown in attachment culture. The surgical treatment cost is high, and the advanced vitiligo and scar constitution are contraindications and have limitation.

The western medicine treatment method has certain limitations for treating the leucoderma, and has poor treatment effect, low cure rate and more adverse reactions.

The traditional Chinese medicine accumulates abundant clinical theories and experiences in the aspect of preventing and treating leucoderma, in ancient books of traditional Chinese medicine, the leucoderma has the names of leucoderma, white barge wind, mottle and the like, and the etiology and pathogenesis of the leucoderma in ancient books of the past are discussed more and mainly divided into 3 types: (1) the disharmony between qi and blood: namely, the invasion of wind evil is considered to be struggled with the skin to cause local qi and blood disharmony of the skin; (2) theory of blood stasis: that is, exogenous pathogenic qi is stagnated in the skin, which leads to qi stagnation and blood stasis and malnutrition of the skin for a long time; (3) treatment from cold: it is considered that cold pathogen invades the exterior to cause qi movement to converge, and meridian and tendons to contract, so that qi, blood and body fluids are coagulated locally on the epidermis, and finally the skin is not good and white spots are generated. The traditional Chinese medicine holds that the leucoderma is caused by the disharmony of the liver, spleen and kidney, and the skin cannot be nourished by qi and blood due to the attack of wind evil on the skin or deficiency or stasis. The modern Chinese medicine treatment mainly comprises a medicinal treatment and a non-medicinal treatment, and the Chinese medicine external treatment is combined treatment on the basis of dialectical treatment, so that the effective rate and the cure rate of the leucoderma are improved. The traditional Chinese medicine adopts the main scheme of the Baigeng pill, the Xiaobaitang and the like, and the total effective rate is 81.63% (the cure rate is 13.9%, and the adverse reaction incidence rate is 18.37%). Wherein Uygur medicine adopts compound Vernonia anthelmintica pill, Xiaobai ointment, etc. as main scheme, and has total effective rate of 82.4% (cure rate of 11.4%, adverse reaction rate of 10%).

Vernonia anthelmintica injection (Chinese medicine standard Z20063652) has the functions of curing, removing mucus, warming skin and coloring, and is clinically used for treating leucoderma, and the main active ingredient of the injection is flavonoid compounds. However, the traditional Chinese medicine prescription is a compound mixture, the dose-effect relationship of the medicine is not clear, the action mechanism of the traditional Chinese medicine is not clear, and the quality standard of the traditional Chinese medicine is yet to be unified. The flavonoids are compounds widely existing in the nature and are distributed in higher plants. Mainly exists in plants of Betulaceae, Rutaceae, Lauraceae, Labiatae, Photiniaceae, Scrophulariaceae, Leguminosae, Gesneriaceae, Rhododendron, Compositae, etc. According to related researches, the flavonoid compound has the effects of resisting oxidation, inflammation, cancer, vascular proliferation and virus.

At present, relatively few reports on related patent documents of flavonoid compounds for preparing anti-leucoderma medicaments exist, and the flavonoid compounds mainly relate to the effect of a specific flavonoid compound and a derivative thereof on the aspect of inducing melanin accumulation. For example, CN201310275882.8 discloses the use of galangin derivatives, namely flavonoids, in the preparation of drugs for preventing and treating vitiligo. The patent mainly describes that galangin derivatives obtained by synthesis methods such as functional group conversion, esterification and the like have the function of promoting melanin accumulation. The invention patent CN201210045334.1 'Vernonia anthelmintica flavone component and a preparation method and application thereof' discloses seven flavone components such as butin, butin and the like separated from Vernonia anthelmintica, and finds that the compounds have the function of treating leucoderma, but the superiority of the function of the flavone with methoxyl group is not clear, and the invention patent only relates to migration of melanocytes, namely accumulation of the melanocytes. CN200410091322.8 found that butin and its derivatives can improve tyrosinase activity, proliferate melanocytes, and increase melanin content in the preparation of drugs for treating pigment metabolism diseases, especially can be used as drugs for treating pigment metabolism diseases such as vitiligo and canities, and there is no new application of methoxyl flavonoids in the preparation of anti-vitiligo drugs.

Relevant researches show that the flavonoid compound with the methoxy functional group (-OCH3) has higher stability in metabolism than the flavonoid compound without the methoxy functional group, and the methoxy flavonoid compound has the best effect of promoting melanogenesis. Therefore, the methoxy flavonoid compound is likely to become a new lead compound for treating the leucoderma.

TABLE 1 existing methods for the treatment of vitiligo

Disclosure of Invention

The invention aims to provide a new application of methoxy flavonoid compounds in preparing anti-vitiligo medicines, namely the methoxy flavonoid compounds with 2-phenylchromone mother nucleus, wherein R1, R2 and R3 contain one or more methoxy groups, and the structure of the methoxy flavonoid compounds is shown in (1). A representative compound is Kaempferide (Kaempferide), and the molecular formula is as follows: c₁₆H₁₂O₆Molecular weight: 300.26, R1 is methoxyl, R2 is hydrogen, R3 is hydroxyl, isorhamnetin (Isooliquitinigenin), molecular formula: c₁₆H₁₂O₇Molecular weight: 316.26, R1 is hydroxyl, R2 is methoxyl, R3 is hydroxyl, Kaempferol 3, 4' -dimethyl ether (Ermanin), molecular formula: c₁₇H₁₄O₆Molecular weight: 314.29, R1 is methoxy, R2 is hydrogen, R3 is methoxy, etc. The methoxy flavonoid compound provided by the invention is not limited to kaempferide, isorhamnetin and kaempferol 3, 4' -dimethyl ether, and comprises all compounds with methoxy flavonoid parent nucleus.

The methoxy flavonoid compounds with 2-phenylchromone parent nucleus are all the methoxy flavonoid compounds, wherein the positions R1, R2 and the like have better methoxy substitution effects, and the methoxy substituted flavonoid compound with the position R1 has the best effect of promoting melanin generation. In addition, the invention provides a pharmaceutical composition or preparation for resisting vitiligo, which uses methoxyflavone or pharmaceutically acceptable sodium salt, potassium salt, solvate or hydrate thereof as an active ingredient.

According to the invention, the pharmaceutical composition or preparation for preventing and treating vitiligo takes methoxyflavone with a treatment effective amount as a main active ingredient, and also comprises one or more pharmaceutically acceptable carriers when needed. The "prevention and treatment effective concentration" refers to the administration concentration of methoxyflavone which can achieve the prevention and treatment effects, and can be different according to the dosage form, the use of a carrier, possible combination with other therapeutic agents and the like.

Pharmaceutically acceptable carriers of the present invention include, but are not limited to: excipients, such as starch and its derivatives, dextrin, calcium hydrogen phosphate, magnesium stearate, microsilica, etc.; disintegrants, such as sodium carboxymethyl cellulose, hydroxypropyl cellulose, and the like; lubricants, such as magnesium stearate and the like; sugar-coating materials such as sucrose, pulvis Talci, gelatin, pigment, and Cera chinensis; film coating materials, such as gastric-soluble film, enteric-coated film, etc. The preparation includes, but is not limited to, injection, tablet, powder, granule, capsule, oral liquid, paste, etc. The flavonoid compound with the methoxyl substitution has higher liver metabolic stability and better gastrointestinal tract absorbability compared with the flavonoid compound without the methoxyl substitution, and is more suitable for being prepared into oral preparations.

The representative methoxy flavonoid compound kaempferide is 2-128 mu M, preferably 32 mu M, the best isorhamnetin is 2-128 mu M, preferably 32 mu M, the best kaempferol is 4-64 mu M, preferably 16 mu M.

The kaempferide and isorhamnetin disclosed by the invention promote the expression of an early differentiation marker miff and a late differentiation marker dct of melanocytes by up-regulating the expression of mc1r and mitf mRNA levels, so as to promote the regeneration and regeneration of the melanocytes. Further research shows that kaempferide and isorhamnetin can up-regulate tyr, tyrp1 and dct mRNA expression, up-regulate melanin steady-state related protein adcy6, weaken melanin metabolism, maintain melanin steady state, and down-regulate glutathione to synthesize gclm, gclc and other related gene expressions, so that more melanin is accumulated, and the function of treating leucoderma is exerted. The kaempferol 3, 4' -dimethyl ether has the related effects of kaempferide and isorhamnetin, can remarkably promote the expression of miff, tyr and dct genes and is dose-dependent, and simultaneously can reduce the expression of mc1r gene, and can also induce the increase of the content of melanin in cells caused by the increase of the active oxygen level of physiological dose in the melanocytes. In addition, Kaempferol 3, 4' -dimethyl ether promotes the expression level of CREB and p-CREB proteins, which shows that Erm promotes melanogenesis by promoting CREB phosphorylation and MITF expression.

The invention has the following beneficial effects: the invention provides a new application of methoxy flavonoid compounds, namely a new application in preparing medicines for resisting leucoderma diseases. The existing vitiligo treatment schemes are all single strategies, namely inducing melanin accumulation. The invention provides a multi-strategy treatment scheme, which can reduce the induction death of oxidative stress on melanocytes and protect the melanocytes while inducing the accumulation of melanin. Inducing the generation of melanocyte and further promoting the production of melanin. Meanwhile, the apoptosis decomposition of the original melanocyte is reduced. Methoxy flavone compounds represented by kaempferide, isorhamnetin, kaempferol 3, 4' -dimethyl ether and the like can remarkably promote the neogenesis and regeneration of melanocytes, cause melanin deposition in a focus area, and simultaneously can up-regulate melanin steady-state related genes adcy6, reduce the decomposition of melanin, maintain the melanin steady state, further induce the further accumulation of melanin, thereby generating the effect of resisting leucoderma. In addition, kaempferide can induce the increase of active oxygen under physiological dosage and reduce the level of glutathione to promote the generation of melanin, can obviously reduce the expression of glutathione synthesis key genes gclm, gclc, ggt1 and gsr, and has a dose-related trend, and the generation of melanin is obviously increased along with the reduction of the glutathione synthesis key genes. The death rate of kaempferide, isorhamnetin and kaempferol 3, 4' -dimethyl ether in the using process is less than 5%, and adverse reactions are avoided, so that the application prospect is good.

Compared with alpha-MSH, the methoxy flavonoid compound has better melanin generation promoting effect and higher cost-effectiveness ratio. The 8-methoxypsoralen is a medicament for clinically treating leucoderma, and methoxy flavonoid compounds provided by the invention have better melanin generation promoting effect than 8-methoxypsoralen, wherein the representative medicaments of the methoxy flavonoid compounds are kaempferide, isorhamnetin and kaempferol 3, 4' -dimethyl ether.

And secondly, the methoxyl flavonoid compound is combined with an oxygen-assisting agent such as butanethionine-sulfoximine, so that the action effect of the methoxyl flavonoid compound is enhanced. Under the simultaneous action of inhibitors of melanin generation related proteins such as mitf, tyrosinase and the like, the methoxy flavonoid compounds eliminate the action of the inhibitors and promote the generation of melanin.

In the invention, the methoxy flavone compound is used for the anti-vitiligo pharmaceutical composition or preparation which takes methoxy flavone or pharmaceutically acceptable salts, solvates or hydrates thereof such as sodium salt, potassium salt and the like as active ingredients. The preparation comprises preparations such as but not limited to liniment, lotion, transdermal preparation, smearing preparation, injection, tablet, powder, granule, capsule, oral liquid, paste and the like, and has various dosage forms, easy preparation, various administration ways and more convenient use.

Finally, compared with the flavonoid compound without methoxy substitution, the flavonoid compound with methoxy substitution has higher liver metabolic stability and better gastrointestinal tract absorbability, and is more suitable for being prepared into an oral preparation which is an optimal preparation form, convenient to take and high in bioavailability.

Drawings

FIG. 1 shows the effect of kaempferol, isorhamnetin, on melanin regeneration in the depigmenting model of adult zebrafish;

FIG. 2 shows the effect of kaempferol, isorhamnetin on melanin content in depigmentation model of adult zebrafish;

FIG. 3 the effect of kaempferol, isorhamnetin, on the production of melanin in neogenetic tissue in a zebrafish tail fin regeneration model;

FIG. 4 shows the effect of kaempferide and isorhamnetin on melanin regeneration and embryo survival rate of young zebra fish;

FIG. 5 shows the effect of kaempferide, isorhamnetin on mRNA levels of genes involved in melanin synthesis in young zebrafish;

FIG. 6 the effect of kaempferide on melanogenesis in adult and juvenile zebrafish;

FIG. 7 Effect of Kaempferide on melanin in the neonatal (primary) and regenerative (B16F10) pigment models of C57BL/6 mice;

FIG. 8 shows the effect of kaempferide on melanin in different cells after pretreatment with MITF and CREB inhibitors;

FIG. 9 effect of kaempferol on expression of glutathione key gene mRNA levels in a cell model;

FIG. 10 the effect of Kaempferol 3, 4' -dimethylether on tyrosinase activity in B16F10 cells;

FIG. 11 Effect of Kaempferol 3, 4' -dimethylether on intracellular and extracellular pigments of C57BL/6 mouse primary cells and B16F 10;

FIG. 12 is a graph showing the effect of Kaempferol 3, 4' -dimethyl ether on melanogenesis in zebrafish juvenile fish;

FIG. 13 the effect of Kaempferol 3, 4' -dimethyl ether on melanin production in vitro model;

Detailed Description

The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto: the methods described in the following examples are not specifically described, and are conventional methods or methods suggested in the specification.

Animals:

purchased from the animal research center in the autonomous region of Uygur autonomous region of Xinjiang, license number: SCXK (New) 2011-

From Yashunity Biotech Co., Ltd, Nanjing, B16F10 melanoma cell line was purchased from cancer cell Bank (Shanghai cell Bank, China, RRID: CVCL _0159)

Reagent:

the kaempferide (purity 98%) is purchased from Shanghai-sourced leaf biotechnology, Inc., the isorhamnetin (purity 98%) is purchased from Shanghai-sourced leaf biotechnology, Inc., and the Kaempferol 3, 4' -dimethyl ether (purity is more than or equal to 98%) is purchased from Shanghai-sourced leaf biotechnology, Inc. DMSO (99.9% pure) was purchased from Beijing Soilebao Tech technologies, Inc., and New copper reagent (98% pure) was purchased from Sigma, USA.

Example 1:

the experimental materials and animals are as specified above.

The names and processing conditions of each group in this embodiment are as follows: (1) blank control (Ctrl); (2) methoxsalen positive drug group (8-MOP, 25 mu mol/L); (3) kaempferol group (2-64 μmol/L); (4) isorhamnetin group (4-64. mu. mol/L). Culturing in water at 24-28 deg.C under light for 14 hr per day, and in dark for 10 hr. The specific treatment is as follows:

100 adult zebra fishes of 4-12 months old are taken and randomly divided into 4 groups of 25 fishes. Control groups were used: treating with 550nmol/L new copper reagent for 20 days, and naturally recovering melanin for 3 days; positive control group: treating with 550nmol/L new copper reagent for 20 days, and treating with 25 μmol/L methoxsalen (8-MOP) for 3 days; isorhamnetin group: treating with 550nmol/L new copper reagent for 20 days, and treating with isorhamnetin (Ire) with different concentrations for 3 days; kaempferol group: 550nmol/L of fresh copper reagent for 20 days, and different concentrations of kaempferol (Kpd) for 3 days. The skin melanin regeneration observation is carried out by using a body type dissecting mirror. Frozen tissue sections of the tissue of the tail of the zebra fish are taken, the tissue is stained by toluidine blue staining solution, and the change of mature melanosomes and melanin ribbons of the skin cross section of the tail of the zebra fish is observed by an optical microscope, and the result is shown in figure 1.

Example 2:

100 adult zebra fishes of 4-12 months old are taken and randomly divided into 4 groups of 25 fishes. Control groups were used: treating with 550nmol/L new copper reagent for 20 days, and naturally recovering melanin for 3 days; positive control group: treating with 550nmol/L new copper reagent for 20 days, and treating with 25 μmol/L methoxsalen (8-MOP) for 3 days; isorhamnetin group: treating with 550nmol/L new copper reagent for 20 days, and treating with isorhamnetin (Ire) with different concentrations for 3 days; kaempferol group: 550nmol/L of fresh copper reagent for 20 days, and different concentrations of kaempferol (Kpd) for 3 days.

6 skin strips of the zebra fish per group were taken, homogenized and then placed in a water bath at 80 ℃ for 2h using a NaOH solution (2mol/L) containing 10% DMSO. Centrifuging to collect supernatant, and obtaining absorbance value (OD) at 350nm_350nm). OD350nm values were calculated per 25mg of skin compared to the control group, and the formula was as follows: ((OD)_350nm/25mg)-(OD_{350nm (control group)}/25mg)/OD_{350nm (control group)}The results are shown in FIG. 2.

Example 3:

the names and processing conditions of each group in this embodiment are as follows:

(1) blank control (Ctrl); (2) methoxsalen positive drug group (8-MOP, 25 mu mol/L); (3) kaempferol group (2-64 μmol/L); (4) isorhamnetin group (4-64. mu. mol/L). Culturing in water at 24-28 deg.C under light for 14 hr per day, and in dark for 10 hr. Taking 4-12 months old zebra fish, cutting off the tail fin in a beveling way, treating the zebra fish for 10 days by using kaempferide, isorhamnetin and methoxsalen respectively, taking the newborn primordium at the tail of the zebra fish to transversely freeze a tissue slice, and staining by using hematoxylin staining solution, wherein the result is shown in figure 3.

Example 4:

(1) blank control (Ctrl); (2) methoxsalen positive drug group (8-MOP, 25 mu mol/L); (3) kaempferol group (2-64 μmol/L); (4) isorhamnetin group (4-64. mu. mol/L). Taking 25-30 zebrafish embryos per group, administering 25 mu mol/L methoxsalen, 32 mu mol/L isorhamnetin and 32 mu mol/L kaempferide 6 hours (6hpf) after fertilization, recording the melanin generation process by using an optical microscope after treating for 72 hours, and counting the survival rate of the embryos, wherein the result is shown in figure 4.

Example 5:

(1) blank control (Ctrl); (2) methoxsalen positive drug group (8-MOP, 25 mu mol/L); (3) kaempferol group (2-64 μmol/L); (4) isorhamnetin group (4-64. mu. mol/L). After treating for 72h, extracting RNA of the zebra fish juvenile fish, and detecting the purity and the concentration by using Q5000; conversing the cDNA into cDNA by using a cDNA reversion kit, and detecting the purity and the concentration by using Q5000; 2Taq PCR Mix is used for amplification, the amplification condition is 38 cycles, the temperature is 95 ℃, and the time is 5 min; ② 95 ℃ for 30 s; ③ 60 ℃ and 30 s; fourthly, the temperature is 72 ℃ for 40 s; fifthly, the temperature is 72 ℃ and the time is 5 min. Products were detected by RT-PCR gel electrophoresis and subjected to grey value analysis, the results are shown in FIG. 5.

Example 6:

according to the direct cause of the leucoderma, a new copper reagent (550nmol/L) of a copper ion chelating agent is used for acting on adult zebra fish for 20 days to enable the tail part of the adult zebra fish to lose melanin, and the clinical leucoderma depigmentation representation is simulated. 100 adult zebra fish strips which are successfully molded are taken and randomly divided into 5 groups, and each group comprises 20 adult zebra fish strips. Control groups were used: treating with 550nmol/L new copper reagent for 20 days, and naturally recovering melanin for 3 days; positive control group: treating with 550nmol/L new copper reagent for 20 days, and treating with 25 μmol/L methoxsalen (8-MOP) for 3 days; kaempferol low concentration group: 550nmol/L of fresh copper reagent for 20 days, and 8. mu. mol/L of kaempferide (Kpd) for 3 days. Concentration group in kaempferide: 550nmol/L of fresh copper reagent for 20 days, and 16. mu. mol/L of kaempferide (Kpd) for 3 days. Kaempferol high concentration group: 550nmol/L of fresh copper reagent for 20 days, 32. mu. mol/L of kaempferide (Kpd) for 3 days. Skin melanin regeneration observation was performed using a stereoscopic dissector (8 ×). Frozen tissue sections of the tissue of the tail of the zebra fish were taken, stained with toluidine blue stain, and the change of mature melanosomes and black pigment bands in the skin cross section of the tail of the zebra fish was observed with an optical microscope (200 ×), and the result is shown in fig. 6.

Example 7:

B16F10 cells were cultured in DMEM containing 10% fetal bovine serum and 1% penicillin/streptomycin in a 5% C02 humidified incubator at 37 ℃. Taking the dorsal skin of a C57BL/6J mouse in a newborn day, soaking for 14-16h under the action of neutral protease II, then separating the epidermis and the dermis, shearing the epidermis as much as possible, soaking the epidermis into trypsin, digesting for 15-20min under the conditions of 37 ℃ and 5% C02, adding a culture medium with the same amount as the trypsin to stop digestion, filtering the cells by using a 0.22 mu m disposable multifunctional filter sieve, centrifuging for 5min at the rotating speed of 800rpm, discarding the supernatant, adding a DMEM complete culture medium containing 10% fetal calf serum and 1% penicillin/streptomycin, and replacing a melanocyte culture medium (MelM) for culture after the cells are completely attached to the wall. B16F10 cells and C57BL/6J cells were treated with media containing methoxsalen and kaempferol at various concentrations for 24 hours. NaOH (2mol/L) containing 10% DMSO was added to each well to lyse the cells. The cells were heated at 80 ℃ for 1 hour and then centrifuged. The content of melanin in the supernatant was determined by spectrophotometry at 405nm, and the results are shown in FIG. 7.

Example 8:

the B16F10 cells and C57BL/6J cells were divided into 6 groups and each was treated with the corresponding drug medium for 24 hours. (1) Blank control (Ctrl); (2) kaempferol group (2-128. mu.M); (3) MITF inhibitor ML 329 group; (4) CREB inhibitors group 666-15; (5) a combined drug group of an MITF inhibitor ML 329 group and kaempferide; (6) CREB inhibitor group 666-15 and kaempferide union, NaOH (2mol/L) containing 10% DMSO was added to each well to lyse the cells. Every 24 hours, melanocyte morphology was observed under a microscope.

Measurement of melanin content of cells, B16F10 cells and primary C57BL/6J mouse epidermal melanocytes were cultured on six-well culture plates and incubated. The cells were treated with a medium containing 8-MOP and kaempferol at various concentrations for 24 hours, and the morphology of the cells was observed under a microscope. NaOH (2mol/L) containing 10% DMSO was added to each well to lyse the cells. The cells were heated at 80 ℃ for 1 hour and then centrifuged. The content of melanin in the supernatant was measured spectrophotometrically at 405nm, and the results are shown in FIG. 8.

Example 9

B16F10 cells and primary C57BL/6J mouse epidermal melanocytes were cultured in six-well culture plates and incubated. Cells were treated with media containing 8-MOP and varying concentrations of kaempferol for 24 hours. NaOH (2mol/L) containing 10% DMSO was added to each well to lyse the cells. Total cellular RNA was isolated using a commercial kit. RNA quality was tested using A260/A280 ratio and 1.5% agarose gel electrophoresis. cDNA Synthesis was performed using Moloney murine leukemia virus reverse transcriptase and the First Strand cDNA Synthesis Kit (Thermo Fisher, USA). Conventional RT-PCR was performed using reagents for Taq enzyme and TIANGEN. The amount of mRNA in the sample was quantified using a SYBR Green-based Rotor-Gene Q (Qiagen, German), and quantified using the 2- Δ Δ Ct method. mRNA expression was normalized using GAPDH as an endogenous control. Amplification was performed for 40 cycles (denaturation at 95 ℃ for 10 min, annealing at 95 ℃ for 5s, and extension at 60 ℃ for 45 s). The glutathione-related protein primer is synthesized by Sanjiangsu Co., Ltd (Shanghai, China). All experiments were repeated at least twice, and the results are shown in FIG. 9

Example 10:

after treating the cells with 4, 8, 16. mu.M Kaempferol 3, 4' -dimethyl ether and two positive drugs 8-MOP 20. mu.M and. alpha. -MSH 200nM for 24 hours, they were washed twice with cold PBS, lysed with 50mM phosphate buffer (pH 6.8) containing 1% Triton X-100, and frozen at 80 ℃ for 60 minutes, and then centrifuged at 15,000rpm for 20 minutes. 150 μ L of protein solution was collected in centrifuge tubes and the amount of protein in each lysate was quantified using the Bradford Standard Assay (BSA). 15mM L-DOPA was added to 20. mu.L of the lysate, and the reaction product was incubated at 37 ℃ for 30min, and the absorbance of the dopachrome was measured at 475 nm. Tyrosinase activity was evaluated as the rate of oxidation of L-3, 4-dihydroxyphenylalanine (L-DOPA), and the results are shown in FIG. 10.

Example 11:

B16F10 and C57BL/6 mouse primary cells were plated at 1.5X 10 per well⁵Cells were cultured overnight in 6-well plates and treated with different concentrations of kaempferol 3, 4' -dimethyl ether (4, 8, 16 μ M) for 24 hours. 8-MOP (20. mu.M) and α -MSH (200nM) were used as positive controls. Cells and supernatant were collected separately. In an extracellular assay, 400. mu.M of 2- [4- (2-hydroxyethyl) piperazin-1-yl]Ethanesulfonic acid buffer (pH 6.8) and EtOH (9: 1, v/v) were added to 1mL of the medium, respectively, at 1mL each. Optical Density (OD) was read at 405 nm. To measure the intracellular melanin content, the collected cell pellets were washed twice in ice-cold PBS and dissolved in 1mol/L NaOH containing 10% DMSO at 80 ℃ for 1 hour. Then, the absorbance was measured at 405 nm. The melanin content is expressed as the ratio of the experimental group to the control group (percentage of the control group), and the results are shown in fig. 11.

Example 12:

(1) blank control (Ctrl); (2) methoxsalen positive drug group (8-MOP, 20 mu mol/L); (3) group α -MSH (200 nmol/L); (4) kaempferol 3, 4' -dimethyl ether (4-64. mu. mol/L). 25-30 zebra fish embryos are taken per group, young zebra fish 1 day after fertilization are decolorized for 1 day by PTU, then treated for 4 days by Kaempferol 3, 4' -dimethyl ether, 8-MOP and alpha-MSH respectively, the expression condition of melanin is observed, the survival rate is counted, and the result is shown in figure 12.

Example 13:

and C57BL/6 mouse skin cells within 1 day of birth are taken to establish a mouse primary melanocyte line. Specifically, the skin of the back of the mouse was taken with ophthalmic scissors and tweezers and the subcutaneous tissue of the mouse was removed as much as possible, placed in a PBS buffer for 1min, then placed in a petri dish containing a Dispase II (5mg/ml) solution in the order of epidermis on top and dermis on a4 ℃ freezer, after 14-16h, the epidermis was gently peeled off from the dermis and cut as much as possible, digested with 0.25% trypsin at 37 ℃ for 15-20min, an equal amount of medium was added to terminate the digestion, the cells were filtered with a 0.22 μm disposable filter sieve, centrifuged at 800rpm for 5min, and the supernatant was removed. Resuspending the cell pellet in DMEM medium containing 10% fetal calf serum, and replacing and supplementing the cell pellet after the cell adheres to the wall the next dayMelanocyte medium (MELM) culture with 1% MelGS. Primary cells were plated at 1.5X 10 per well⁵Cells were cultured overnight in 6-well plates and treated with kaempferol 3, 4' -dimethyl ether (4, 8, 16 μ M) at the indicated concentrations for 24 hours. 8-MOP (20. mu.M) and α -MSH (200nM) were used as positive controls. Cells and supernatant were collected separately. In an extracellular assay, 400. mu.M of 2- [4- (2-hydroxyethyl) piperazin-1-yl]Ethanesulfonic acid buffer (pH 6.8) and EtOH (9: 1, v/v) were added to 1mL of the medium, respectively, at 1mL each. Optical Density (OD) was read at 405 nm. To measure the intracellular melanin content, the collected cell pellets were washed twice in ice-cold PBS and dissolved in 1mol/L NaOH containing 10% DMSO at 80 ℃ for 1 hour. Then, the absorbance was measured at 405 nm. Morphological observation of primary cells, photographing of cell pellet and measurement of relative melanin content inside and outside cells, the melanin content is expressed as the ratio of experimental group to control group (percentage of control group), and the results are shown in fig. 13.

Example 14:

B16F10 melanoma cells were cultured at 1X 10⁴Cells/ml were seeded overnight in 24-well plates and then treated with kaempferol (0-128 μ M) for 96 hours, with cell morphology observed and cell viability measured by MTT every 24 hours. Take 100. mu.L of 5X 10⁴The cells per mL are inoculated in a 96-well plate, put into a cell culture box at 37 ℃ overnight, and treated with kaempferide, isorhamnetin and kaempferol 3, 4' -dimethyl ether at different concentrations (0, 2, 4, 8, 16, 32 and 64 mu M) for 24 h. mu.L of 5mg/mL MTT solution was added to each well and incubated for 4 hours. After discarding the medium, the resulting formazan was dissolved in 150 μ L DMSO, and then 96-well plates were placed on a plate shaker for 10 minutes with shaking, and absorbance values were measured at 570nm using a Thermo 3001 plate reader. Cell viability was calculated as follows: (administration group OD-blank OD)/(control group OD-blank OD). Cells were collected and stained with Muse cell viability assay kit for 10 minutes. The dead cell population was analyzed by flow cytometry and the rate of melanocyte apoptosis was decreased by kaempferide-treated group.

Example 15:

(1) blank control (Ctrl); (2) kaempferol group (2-128. mu.M); (3) MITF inhibitor ML 329 group; (4) CREB inhibitors group 666-15; (5) a combined drug group of an MITF inhibitor ML 329 group and kaempferide; (6) CREB inhibitor 666-15 group and kaempferide combination group. Taking 25-30 zebra fish embryos per group, administering kaempferide and MITF inhibitors ML 329 and CREB inhibitors 666-15 with corresponding concentrations 6 hours (6hpf) after fertilization, recording melanin generation process by using an optical microscope after treating for 72 hours, and counting the embryo survival rate.

Example 16:

the B16F10 cells and C57BL/6J cells were divided into 6 groups and each was treated with the corresponding drug medium for 24 hours. (1) Blank control (Ctrl); (2) kaempferol group (2-128. mu.M); (3) ADCY6 inhibitor SQ 22536; (4) a combination of an ADCY6 inhibitor SQ22536 and kaempferide; NaOH (2N) containing 10% DMSO was added to each well to lyse the cells. Every 24 hours, melanocyte morphology was observed under a microscope.

Measurement of melanin content of cells, B16F10 cells and primary C57BL/6J mouse epidermal melanocytes were cultured on six-well culture plates and incubated. The cells were treated with a medium containing 8-MOP and various concentrations of kaempferide for 24 hours, and the morphology of the cells was observed under a microscope. NaOH (2mol/L) containing 10% DMSO was added to each well to lyse the cells. The cells were heated at 80 ℃ for 1 hour and then centrifuged. The content of melanin in the supernatant was determined spectrophotometrically at 405 nm.

Example 17:

detecting the specific absorbance (A490) value of a reaction product at 490nm by using an enzyme-linked immunosorbent assay (ELISA) detector by adopting a tyrosinase dopa rate oxidation method, carrying out reaction in a 96-well culture plate, wherein the total reaction system is 200 mu L, and Phenylthiourea (PTU) (100 nm) is used as a positive control: wherein, 2 mu L of kaempferol with different concentrations of a test sample is mixed with 60 mu L of 100mM phosphate buffer, 40 mu L of 210U/ml tyrosinase and 100 mu L of 1.5mM L-tyrosine, after the sample is added, a 96-hole culture plate is placed in a water bath box with the temperature of 37 ℃ for incubation for 20min, the A490 value of each hole and three-time holes of each concentration are measured under a microplate reader, and finally the activation rate of the tyrosinase is calculated. The values for each measurement are expressed as a percentage change relative to the untreated control (reaction mixture without kaempferol). Groups were treated with kaempferol diluted in buffer without tyrosinase, excluding color interference in absorbance measurements.

Example 18:

cells were treated at 5X 10³The density of each well was plated in 96-well plates and incubated overnight at 37 ℃. The corresponding drugs were added in groups of (1) blank control (Ctrl); (2) kaempferol group (4-16. mu.M) after further 2h incubation of the cells, 20. mu.M 2 ', 7' -dichlorofluorescent xanthate diacetate (DCFH-DA) or 10. mu.M fluorescein diacetate (CMFDA) was added and incubated for 40 min. ROS in cells were relatively quantified by DCFH-DA, which was hydrolyzed by intracellular esterases to DCFH, and then rapidly oxidized to generate the strongly fluorescent product 2 ', 7' -Dichlorofluorescein (DCF), i.e. ROS levels were measured indirectly by oxidation of non-fluorescent DCFHDA to fluorescent DCF, and fluorescence values were read using a microplate reader (Thermo variaskan Flash 3001, USA) at 485nm excitation and 535nm emission wavelengths. Lipophilic groups in CMFDA can be hydrolyzed by intracytoplasmic nonspecific esterase to generate 5-chloromethyl fluorescein, green fluorescence can be emitted, the green fluorescence and glutathione in intracellular protein and polypeptide can generate adduct under the action of glutathione mercaptotransferase, and the fluorescence value of the adduct is analyzed at an excitation wavelength of 522nm and an emission wavelength of Em ═ 595 nm. Changes in ermannin induced ROS and GSH levels were also recorded at 200-fold magnification by using a fluorescence microscope (Axio Observer, Zeiss, germany).

The embodiment shows that the flavonoids, particularly methoxy flavonoids kaempferide and isorhamnetin, promote the expression of an early differentiation marker miff and a late differentiation marker dct of melanocytes by up-regulating the mRNA level expression of mc1r and mitf, and further promote the regeneration of the melanocytes. Further research shows that kaempferide up-regulates tyr, tyrp1 and dct mRNA expression, and kaempferide up-regulates melanin homeostasis-related protein adcy6, so that melanin metabolism is weakened, more melanin is accumulated, and the effect of treating leucoderma is exerted. The increase of the active oxygen level in the melanocyte caused by the kaempferol 3, 4' -dimethyl ether can cause the increase of the melanin content in the cell, and the effect of promoting the increase of the melanin content by using the pro-oxidant is better. Meanwhile, kaempferol 3, 4 '-dimethyl ether can remarkably promote the expression of miff, tyr and dct genes and is dose-dependent, the expression of mc1r gene is reduced, and kaempferol 3, 4' -dimethyl ether promotes the expression level of CREB and p-CREB proteins, which shows that Erm promotes melanogenesis by promoting CREB phosphorylation and MITF expression. The methoxy flavonoid compound can improve the vitiligo model lesion, has no adverse reaction and high survival rate, and can be used for preparing anti-vitiligo medicaments.

Although the invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that many modifications and improvements can be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims

1. The application of the methoxy flavonoid compound in preparing the anti-vitiligo medicine is characterized in that: methoxy flavonoids with a 2-phenylchromone nucleus, i.e. R₁、R₂、R₃Containing one or more methoxy groups, with R₁The compound is Kaempferide (Kaempferide) R with optimal effect on methoxyl₁Is methoxy, R₂Is hydrogen, R₃Is hydroxy, isorhamnetin (Isooliquitinigenin) R₁Is hydroxy, R₂Is methoxy, R₃Is hydroxy, Kaempferol 3, 4' -dimethyl ether (Ermanin) R₁Is methoxy, R₂Is hydrogen, R₃Methoxy, etc.

2. The use of methoxy flavonoids according to claim 1 in the preparation of anti-vitiligo drugs, which is characterized in that: the methoxy flavonoid compounds promote the generation of melanin and reduce the decomposition of the melanin to play a role in treating and/or preventing leucoderma, can promote the regeneration and the neogenesis of melanocytes by inducing the expression of genes related to the melanin, inducing the rise of active oxygen under physiological dosage and reducing the level of glutathione to promote the generation of the melanin, promote the recovery of the melanocytes at lesion parts, reduce the apoptosis of the melanocytes, reduce the decomposition of the pigment of the original melanocytes, induce the accumulation of the melanin and maintain the steady state of the melanin.

3. The use of methoxy flavonoids according to claim 1 in the preparation of anti-vitiligo drugs, which is characterized in that: the methoxy flavonoid compound and the oxygen-assisting agent such as buthionine-sulfoximine are combined for use, the effect of the methoxy flavonoid compound is enhanced, and the methoxy flavonoid compound further promotes the generation of melanin under the simultaneous action of melanin generation related protein inhibitors such as Mitf, tyrosinase and the like.

4. The use of methoxy flavonoids according to claims 1, 2 and 3 in the preparation of anti-vitiligo drugs, the methoxy flavonoids mentioned in the present invention represents drugs, the blood concentration of kaempferide in treating vitiligo is 2-128 μ M, preferably 32 μ M for the optimal concentration, and the blood concentration of isorhamnetin in treating vitiligo is 2-128 μ M, preferably 32 μ M for the optimal concentration. The blood concentration of the kaempferol 3, 4' -dimethyl ether in treating the vitiligo is 4-64 mu M, and the optimal concentration is preferably 16 mu M.

5. The use of methoxy flavonoids according to claims 1, 2, 3, 4 in the preparation of anti-vitiligo drugs, characterized in that: the methoxy flavone compound is used for a pharmaceutical composition or preparation for preventing/treating leucoderma by taking methoxy flavone or pharmaceutically acceptable salts, solvates or hydrates such as sodium salt and potassium salt thereof as active ingredients, and the preparation comprises but is not limited to liniment, lotion, a skin-sticking preparation, a smearing preparation, an injection, tablets, powder, granules, capsules, oral liquid, paste and the like, wherein the flavonoid compound with methoxy substitution has higher liver metabolic stability and better gastrointestinal absorption compared with the flavonoid compound without methoxy substitution, and the oral preparation is an optimal dosage form.