Application of alkannin in preparation of drugs for resisting photodermatosis

Abstract

The invention discloses application of alkannin in preparation of drugs for resisting photodermatosis. The shikonin emulsion is used for external treatment of a constructed photoaging mouse model, and results show that after the shikonin emulsion is intervened for one week, mouse epidermis is thickened, namely, the mouse epidermis is recovered to a normal level, and after the shikonin emulsion is intervened for 14 days, mouse true dermatitis cells are also close to the normal level. When the shikonin emulsion intervenes for 7 days, cuticle pigment is reduced, the density and arrangement of dermal collagenous fibers are obviously improved, and after the shikonin emulsion intervenes for 14 days, the dermal collagenous fibers return to a normal state. The invention provides an important experimental basis for the treatment of photoaging by the shikonin emulsion through animal experiments. Furthermore, clinical tests are carried out, and the result shows that the external shikonin emulsion has a remarkable treatment effect on human actinic dermatitis clinically. Therefore, the invention provides a novel therapeutic drug and scheme for prevention and treatment of photodermatosis, and has good clinical application prospects and high clinical application values.

Description

Technical Field

[0001] The invention relates to application of shikonin in preparing a photoresistance dermatopathy drug, belonging to the technical field of biomedicine. Background Art

[0002] Ancient Chinese doctors have a long history of using lithospermum officinale to treat skin diseases. According to the "Wu Er Bing Fang", "Shen Nong's Herbal Classic" and "Compendium of Materia Medica", lithospermum officinale has the effects of promoting blood circulation, cooling blood, and detoxifying, and can be used to treat blood heat and ulcers. The "Compendium of Materia Medica" records that lithospermum officinale can treat "those with sores and rashes that cannot be cured". In terms of external treatment, "Bie Lu" points out that lithospermum officinale ointment can treat children's sores and facial ulcers, and "Yao Xing Lun" records that lithospermum officinale can treat malignant sores, tinea versicolor, macules, and pox.

[0003] Shikonin is the active ingredient of lithospermum officinale extract, including 5,8-dihydroxy-1,4-naphthoquinone, L-shikonin (Akanin), D-shikonin (Shikonin), etc., collectively referred to as shikonin. Previous studies have shown that in addition to its anti-tumor, anti-inflammatory and anti-microbial effects, shikonin is widely used in the field of dermatology. For example, lithospermum officinale oil, lithospermum officinale cream, and lithospermum officinale zinc oxide oil are mainly used to treat skin burns, eczema, and infant diaper dermatitis.

[0004] In the field of medicine, there are more than 1,000 invention patents related to lithospermum extraction methods, medicinal formulas, skin care formulas, etc. Among them, the patents related to dermatology in recent years include: a method for making lithospermum repair cream (application publication number CN 111557856A), an anti-aging and repairing lithospermum composition and its application in skin care products (application publication number CN113712845A), a lithospermum hip diaper (authorization announcement number CN 215273941U), etc. The above studies show that lithospermum has both good anti-inflammatory effects and good skin repair effects, so it can be used in skin care formulas.

[0005] However, no relevant reports have been found on whether shikonin can prevent and treat photodermatosis, especially against photoaging.

[0006] Skin aging is caused by genetic factors (natural aging) and environmental factors. The most important environmental factor is photoaging caused by ultraviolet radiation from sunlight. Photoaging is manifested as loose skin, roughness, wrinkles, dull color, etc. [1] Photoaging and normal aging may have different molecular mechanisms in inducing inflammation and immunosuppression, mainly oxidative stress, inflammatory response, cell apoptosis, cell aging, etc. [2-3]Skin photoaging not only affects the patient's appearance, but also causes skin tumors in some patients. One of the most common ways to prevent photoaging is to use sunscreen, which uses titanium dioxide or zinc oxide to block ultraviolet rays from damaging the skin. However, sunscreen has little effect on repairing skin photoaging that has already occurred. [4] In the treatment of photoaging, retinoic acid, 5-fluorouracil and physical therapy (such as intense pulsed light, fractional laser, picosecond laser, ultrasonic cannon) all have certain effects, but they are prone to cause adverse reactions, thus limiting their widespread application. [5] .

[0007] In recent years, Chinese herbal extracts and their active ingredients have gradually gained attention in the field of preventing and treating skin photoaging due to their few adverse reactions and positive efficacy. Related research mainly focuses on more than 20 Chinese herbal ingredients such as ginseng. [6] .

[0008] References:

[0009] [1]Oh Jin-Su, Lee Sang-Jun, Choung Se-Young. Lithospermum erythrorhizonAlleviates Atopic Dermatitis-like Skin Lesions by Restoring Immune Balance and Skin Barrier Function in 2.4-Dinitrochlorobenzene-Induced NC / NgaMice. Nutrients, 2021, 13(9):3209.

[0010] [2] Liang Hui, Wang Shuya. Effectiveness of gromwell(Lithospermumerythrorhizon)oil in the treatment of toxic epidermal necrolysis in a patient with dermatomyositis. Dermatol Ther, 2020, 33(6):e14067.

[0011] [3] Zhao Bian, ed. Chinese Clinical Dermatology, Jiangsu Phoenix Science and Technology Press, 2nd edition, April 2017. 182-183.

[0012] [4] Salminen A, Kaarniranta K, Kauppinen A. Photoaging: UV radiation-induced inflammation and immunosuppression accelerate the aging process in the skin. InflammRes. 2022, Aug; 71(7-8):817-831.

[0013] [5] KJ, A, R,etal.The impact of ultraviolet radiation on skin photoaging-review of in vitrostudies.J Cosmet Dermatol.2021Nov;20(11):3427-3431.

[0014] [6]Guan LL, Lim HW, Mohammad TF. Sunscreens and Photoaging: A Review ofCurrent Literature. Am J Clin Dermatol. 2021Nov; 22(6):819-828. Summary of the invention

[0015] The purpose of the present invention is to provide application of shikonin in preparing photoresistant dermatopathy drugs.

[0016] In order to achieve the above object, the present invention provides the use of shikonin in preparing an external preparation for photoresistant dermatosis.

[0017] Preferably, the photodermatosis includes photoaging and actinic dermatitis.

[0018] Preferably, the dosage form of the external preparation is selected from any one of emulsion, gel, ointment, spray, solution, film coating and oil.

[0019] Preferably, the external preparation comprises an active pharmaceutical ingredient and a pharmaceutically acceptable carrier and / or excipient, and the active pharmaceutical ingredient is shikonin.

[0020] Preferably, the content of shikonin in the external preparation is 5 mg-100 mg / 100 mL.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The present invention shows that lithospermum emulsion has anti-photoaging effect through animal experiments, which provides an important experimental basis for the lithospermum emulsion to treat photoaging. Furthermore, the present invention conducts clinical trials, and the results show that the external lithospermum emulsion has a significant therapeutic effect on clinical human actinic dermatitis; therefore, the present invention provides a new therapeutic drug and scheme for the prevention and treatment of photodermatosis, and has good clinical application prospects and high clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a photo of a healthy depilated mouse;

[0024] Figure 2 HE staining of healthy mouse skin tissue;

[0025] Figure 3 This is a photo of photoaged mice;

[0026] Figure 4 HE staining of photoaged mouse skin tissue;

[0027] Figure 5 HE staining of healthy mouse skin tissue, with 1.50±0.51 layers of epidermal cells and 1-2 dermal inflammatory cells per field of view;

[0028] Figure 6 This is the HE staining of the skin tissue of the photoaging model mouse. The epidermal cells are 4.93±0.64 layers. The dermal mixed inflammatory cells are 141.39±10.88 / field of view;

[0029] Figure 7 This is the HE staining picture of the skin tissue of the natural recovery group on day 7. The number of epidermal cells is 3.10±0.71 layers; the number of dermal inflammatory cells is 59.60±4.60 / field of view;

[0030] Figure 8 This is the HE staining picture of the skin tissue of the natural recovery group on day 14. The number of epidermal cells is 1.73±0.45 layers; the number of dermal inflammatory cells is 29.37±3.60 per field of view;

[0031] Fig. 9 This is the HE staining picture of the skin tissue after 7 days of matrix intervention. The number of epidermal cells is 2.97±0.61 layers; the number of dermal inflammatory cells is 57.27±5.57 / field of view;

[0032] Fig.10 This is the HE staining picture of the skin tissue after 14 days of matrix intervention. The number of epidermal cells is 2.13±0.43 layers; the number of dermal inflammatory cells is 30.93±7.53 / field of view;

[0033] Fig.11This is the HE staining picture of the skin tissue after 7 days of intervention with Lithospermum officinale emulsion. The number of epidermal layers is 2.03±0.41 layers. The number of dermal inflammatory cells is 24.23±2.90 per field of view.

[0034] Fig.12 This is the HE staining picture of the skin tissue after 14 days of intervention with Lithospermum officinale emulsion. The number of epidermal layers is 1.87±0.63 layers. The number of dermal inflammatory cells is 4.07±0.87 per field of view.

[0035] Fig.13 This is an image of ferrous sulfate staining of healthy mouse skin tissue: the stratum corneum appears light gray without blue-green stripes, and the dermal collagen fibers are arranged in an orderly manner;

[0036] Fig.14 This is an image of ferrous sulfate staining of the skin tissue of photoaging mice before intervention: the stratum corneum appears as a blue-green strip, the epidermis thickens, the dermal collagen fibers break and shorten, and the density increases;

[0037] Fig.15 This is an image of ferrous sulfate staining of skin tissue of photoaged mice after 7 days of natural recovery: the blue-green stripes in the stratum corneum are lighter than before the intervention; the collagen fibers are still similar to those before the intervention;

[0038] Fig.16 This is an image of ferrous sulfate staining of skin tissue of photoaged mice after 14 days of natural recovery: the blue-green stripes in the stratum corneum faded again; the collagen fibers were similar to those before intervention;

[0039] Fig.17 This is an image of ferrous sulfate staining of skin tissue of photoaged mice after 7 days of matrix milk intervention: the stratum corneum has obvious blue-green stripes; the collagen fibers are similar to those before intervention;

[0040] Fig.18 This is an image of ferrous sulfate staining of skin tissue of photoaged mice after 14 days of matrix milk intervention: the blue-green stripes in the stratum corneum faded; the collagen fibers were similar to those before intervention;

[0041] Fig.19 This is an image of ferrous sulfate staining of the skin tissue of photoaged mice after 7 days of intervention with lithospermum officinale milk: the blue-green stripes in the stratum corneum became lighter than before the intervention; the collagen fibers were close to normal morphology;

[0042] Fig. 20 This is an image of ferrous sulfate staining of the skin tissue of photoaged mice after 14 days of intervention with comfrey milk: the blue-green stripes in the stratum corneum disappeared, and the collagen fibers were close to normal morphology. DETAILED DESCRIPTION

[0043] In order to make the present invention more clearly understood, preferred embodiments are described in detail below with reference to the accompanying drawings.

[0044] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0045] Example

[0046] (I) Materials, reagents and instruments

[0047] Kunming mice: female, clean grade, 3 weeks old, weighing 18-20 g (Shanghai Regen Biotechnology Co., Ltd., No.: 2023062614).

[0048] Drugs and reagents: shikonin (Beijing Oriental Miaosen Biotechnology Co., Ltd., batch number: 112585-00025-5045); emulsion base (Guangzhou Boxin Cosmetics Co., Ltd., batch number 2023110105); melanin staining solution (reagent for ferrous sulfate method; batch number: C230801); HE staining reagent set.

[0049] Experimental instruments: 30W ultraviolet lamp with a wavelength of 320nm (Beijing Zhongyi Futeng Technology Co., Ltd.); DHG-9038A electric constant temperature blast drying oven (Shanghai Jinghong Experimental Equipment Co., Ltd.); Leica rotary slicer (model: Histocore Biocut).

[0050] (II) Methods

[0051] Establishment of a mouse photoaging model: There are many types of photoaging animal models, and there is no unified standard (He H, Li A, LiS, et al. Natural components in sunscreens: Topical formulations with sunprotection factor (SPF). Biomed Pharmacother. 2021 Feb; 134: 111161.). This experiment referred to most photoaging experimental models, that is, female Kunming mice of the same batch were used for modeling, and they were adaptively raised for 7 days. The ultraviolet (wavelength 320nm) irradiator was placed 30cm above the mice, with an energy of 14J / cm 2, irradiated for 60 days, irradiated once a day for 2 hours each time in the first 30 days, and irradiated twice a day for 2 hours each time in the last 30 days. When the skin in the depilatory area on the back shows photoaging symptoms such as dryness, desquamation, and wrinkles, the model is successfully established. Grouping of photoaging model mice and its intervention-90 photoaging model mice were randomly divided into 3 groups, 30 in each group, namely the natural recovery group (i.e. positive control group), the matrix group, and the lithospermum group. Among them, the natural recovery group was not treated, the matrix group was applied with emulsion matrix, and the lithospermum group was applied with shikonin emulsion (shikonin: 50 mg + emulsion matrix: 100 mL). According to the unit area conversion of human skin care products for mice, the corresponding emulsion mass for each external application of 3cm×3cm area on the back of each mouse was 75 mg, twice a day.

[0052] Skin tissue pathological examination: routine pathological sections were prepared and stained with HE and ferrous sulfate respectively.

[0053] 3. Statistical analysis

[0054] The experimental data were statistically analyzed using Microsoft EXEL software, the mean was expressed as x±s, and the difference between the two groups was tested using t-test, with P < 0.05 being considered statistically significant.

[0055] (IV) Animal experiments and results

[0056] Healthy mouse skin and photoaged mouse skin lesions - macroscopic observation and histopathological changes: Figure 1 These are healthy hairless mice, with light red skin and a smooth surface as seen with the naked eye. Figure 2 This is a picture of healthy mouse tissue. The epidermis is blue, the epidermal cells are 1-2 layers, the dermal collagen fibers are red and arranged in an orderly wavy manner, hair follicles and sebaceous glands can be seen in the dermis, and the subcutaneous tissue is loose fat lobules and interlobular septa. Figure 3 These are photoaging model mice, and their skin appears dark brown, dry and scaly to the naked eye. Figure 4 This is the histopathological image of photoaged mice. The keratinocytes in the blue epidermis thicken to 4-6 layers, the dermal collagen fibers are broken and shortened, the density increases, and the arrangement is disordered. There are a large number of inflammatory cell infiltrations in the dermis (histiocytes, lymphocytes, neutrophils, eosinophils).

[0057] The histopathological changes of the three groups of photoaged mice at different time points: HE staining was used to observe the number of epidermal cell layers and the number of dermal inflammatory cells / field of view, where dermal inflammatory cells refer to neutrophils, eosinophils, lymphocytes and tissue cells. The ferrous sulfate method was used to observe the changes in the melanin content of the stratum corneum and the arrangement of collagen fibers. The histopathological changes of HE staining before intervention, 7 days after intervention and 14 days after intervention were as follows: Figures 5 to 12 shown.

[0058] The statistical comparison of HE staining results of each group is as follows:

[0059] Natural recovery group: On days 0, 7 and 14, the number of epidermal cell layers was 4.93±0.64, 3.10±0.71 and 1.73±0.45, respectively, indicating that the epidermal layer gradually recovered from thickening to a healthy level; the number of dermal inflammatory cells per field of view was 141.39±10.88, 59.60±4.60 and 29.37±3.60, respectively, indicating that the number of dermal inflammatory cells also gradually decreased, but did not return to normal levels at the end of the experiment.

[0060] Comparison between the natural recovery group and the matrix group: At 7 days of intervention, the comparison of the epidermis between the two groups was t=0.441, P>0.05; the comparison of the number of dermatitis cells between the two groups was t=0.082, P>0.05. At 14 days of intervention, the comparison of the epidermis between the two groups was t=0.001, P>0.05; the comparison of the number of dermatitis cells between the two groups was t=0.308, P>0.05. There was no significant difference in the number of epidermis and dermatitis cells between the two groups, indicating that the matrix emulsion cannot promote the repair of photoaging.

[0061] Comparison between the lithospermum group and the matrix group: at 7 days of intervention, the comparison of the epidermis between the two groups was t=4.369, P<0.01; the comparison of the number of dermatitis cells between the two groups was t=4.594, P<0.01. At 14 days of intervention, the comparison of the epidermis between the two groups was t=0.092, P>0.05; the comparison of the number of dermatitis cells between the two groups was t=4.838, P<0.01. This shows that at the 7th day of intervention with lithospermum emulsion, the number of epidermis layers returned to normal levels, and the number of dermatitis cells was significantly reduced; at 14 days of intervention, the recovery of dermatitis cells in the lithospermum group was significantly better than that in the control group, indicating that lithospermum emulsion can effectively resist the inflammatory cell infiltration of photoaging.

[0062] Experimental results of ferrous sulfate staining of mice in each group:

[0063] Staining principle: Melanin can absorb ferrous ions to form a complex. Ferrous ions combine with potassium ferrocyanide to form a Teng's blue reaction, which dyes melanin blue-green. In addition, VG can be used for re-staining to dye collagen fibers pink.

[0064] Experimental results: (1) Histopathological images of healthy mice and photoaging model mice Figure 13-14 (2) Natural recovery group and matrix group: At 7 and 14 days of intervention, the blue-green stripes in the stratum corneum of each group faded synchronously with time, indicating that melanin production gradually decreased due to the cessation of ultraviolet radiation. In addition, at 7 and 14 days, there was no significant change in the proliferation and breakage of dermal collagen fibers in the matrix group compared with the photoaging model group. Figure 15-18(3) Lithospermum group: On the 7th day of intervention, the green stripes in the stratum corneum were significantly lighter than those in the matrix group; on the 14th day of intervention, the blue-green stripes in the stratum corneum of the Lithospermum group almost disappeared. In addition, on the 7th day of intervention, the collagen fiber morphology of the Lithospermum group was close to that of healthy mice; on the 14th day of intervention, the collagen fiber morphology completely returned to normal, as shown in Figure 2. Figure 19-20 shown.

[0065] The above results show that photoaged mice have the ability to naturally repair epidermal thickening, dermal inflammation and stratum corneum melanin deposition, but the repair of damaged collagen fibers is slower than other indicators. Compared with the natural recovery group, the matrix emulsion intervention group had no obvious advantages in epidermal thickening, dermal inflammation, stratum corneum pigment deposition and collagen fiber hyperplasia and rupture, indicating that a single matrix emulsion cannot prevent and treat photoaging. Compared with other groups, the lithospermum emulsion group showed that epidermal thickening returned to normal levels after 1 week of lithospermum emulsion intervention, and dermal inflammatory cells were close to normal levels after 14 days of intervention. After 7 days of lithospermum emulsion intervention, stratum corneum pigmentation decreased, and the density and arrangement of dermal collagen fibers improved significantly. After 14 days of intervention, dermal collagen fibers returned to normal. The above studies provide important experimental basis for the treatment of photoaging with lithospermum emulsion.

[0066] (V) Clinical trials and results:

[0067] A total of 170 patients with chronic actinic dermatitis in spring and summer were enrolled in the outpatient dermatology department from May 2022 to September 2023. All cases were randomly divided into a treatment group of 85 cases (all patients in the treatment group completed the experiment) and a control group of 85 cases (82 patients in the control group completed the experiment). The gender, age, course of disease, and condition of the two groups of patients were statistically processed, and there was no significant difference (P>0.05), that is, the two groups were comparable.

[0068] Diagnostic criteria, inclusion criteria and exclusion criteria: The diagnosis meets the diagnostic criteria of chronic actinic dermatitis in the Chinese Journal of Clinical Dermatology edited by Zhao Bian (i.e., persistent dermatitis or eczematous skin lesions, mainly affecting the exposed area. The "minimum erythema dose" value of the patient's non-lesional area is significantly lower than that of normal people, and the light patch test or light stimulation test is positive; histopathology shows dermatitis or eczema-like manifestations). Inclusion criteria: No use of photosensitizing drugs or foods within 1 month, and commitment not to use photosensitive foods or drugs during treatment. Exclusion criteria: Oral photosensitizing drugs or; pregnant women and children; patients with severe liver and kidney dysfunction and mental illness.

[0069] Treatment methods and grouping: The treatment group was treated with oral hydroxychloroquine sulfate tablets 0.1g, twice a day, and shikonin emulsion, twice a day. The control group was treated with oral hydroxychloroquine sulfate tablets 0.1g, twice a day, and emulsion matrix, twice a day. The efficacy was evaluated at the end of the trial for 2 weeks. Evaluation was based on the area of ​​lesions, erythema, desquamation, and itching. Cured: clinical symptoms and signs subsided by more than 95%. Significantly effective: symptoms were relieved by 80% to 94%. Improved: symptoms were relieved by 50% to 79%. Ineffective: no improvement in symptoms.

[0070] Results: The efficacy of the two groups is shown in Table 4.

[0071]

[0072] Treatment group: cure rate was 54.12% (46 / 85), marked efficacy was 28.23% (24 / 85), effective rate was 17.65% (15 / 85), ineffective rate was 0% (0 / 85). Control group: cure rate was 31.25% (25 / 80), marked efficacy was 38.75% (31 / 80), effective rate was 30% (24 / 80), ineffective rate was 0% (0 / 80). Comparing the cure rates of the two groups, X 2 =5.849﹥3.841, so P﹤0.05, the difference between the two groups is statistically significant.

[0073] Adverse reaction observation: The blood routine, liver function and kidney function of the two groups of patients were normal before and after treatment. Among the 85 patients in the treatment group, 3 patients had mild pain and discomfort due to contact of shikonin emulsion with the corner of the eye. They were immediately rinsed with tap water without other treatment and returned to normal the next day. No adverse reactions occurred in the 80 patients in the control group.

[0074] Summary: The above preliminary animal experiments and clinical trials show that shikonin emulsion has both an antagonistic effect on the photoaging model of mice and a preventive and therapeutic effect on human actinic dermatitis. Therefore, the present invention provides the use of shikonin in the preparation of anti-sun aging drugs, which is of great significance for the prevention and treatment of photodermatosis, and at the same time opens up a new research direction for the natural plant extract-shikonin to prevent and treat human photodermatosis.

[0075] The above description is only a preferred embodiment of the present invention and is not any formal or substantial limitation of the present invention. It should be pointed out that a person skilled in the art can make several improvements and supplements without departing from the present invention, and these improvements and supplements should also be regarded as the protection scope of the present invention.

Claims

1. Application of shikonin in the preparation of external preparations for photoresistant dermatosis.

2. The use according to claim 1, characterized in that: The photodermatosis includes photoaging and actinic dermatitis.

3. The use according to claim 1, characterized in that: The dosage form of the external preparation is selected from any one of emulsion, gel, ointment, spray, solution, film coating and oil.

4. The use according to claim 1, characterized in that: The external preparation comprises an active pharmaceutical ingredient and a pharmaceutically acceptable carrier and / or excipient, and the active pharmaceutical ingredient is shikonin.

5. The use according to claim 4, characterized in that: The content of shikonin in the external preparation is 5mg-100mg / 100mL.

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