A baicalin external preparation and its application in the treatment of atopic dermatitis

Abstract

The invention discloses a baicalin external preparation and its application in the treatment of atopic dermatitis. The preparation has baicalin as an active ingredient and is prepared into a dosage form that can be applied to the skin and mucous membranes in combination with pharmaceutical excipients. The external preparation can be used for the treatment of atopic dermatitis, has the characteristics of being easy to use and having a significant effect, and has broad application prospects.

Description

Technical Field

[0001] The invention belongs to medical preparations and applications, and specifically relates to a baicalin external preparation and application thereof in the treatment of atopic dermatitis. Background Art

[0002] Atopic dermatitis is a chronic, recurrent inflammatory skin disease, the most basic characteristics of which are dry skin, chronic eczematous skin lesions, and obvious itching. Over the past few decades, the prevalence and incidence of atopic dermatitis have continued to increase. Data show that the prevalence of children worldwide is 15% to 20%, and the prevalence of adults is as high as 10%. Atopic dermatitis has become the 15th most common non-fatal disease and the skin disease with the highest disease burden. According to the medication recommendations of the "Guidelines for the Diagnosis and Treatment of Atopic Dermatitis in China" (2020 edition), drug treatment is divided into topical drug treatment and systemic drug treatment. The first-line medication for topical drug treatment is glucocorticoids, but long-term and large-area use of glucocorticoids can cause adverse reactions to the skin and the system. The maintenance therapy for topical drug treatment is topical anti-inflammatory drugs, such as medium- and weak-effect glucocorticoids or calcineurin inhibitors to reduce recurrences, and its adverse reactions are mainly local burning and irritation. Systemic drug treatment is mainly used for the adjuvant treatment of atopic dermatitis, which is oral antihistamines, immunosuppressants, and glucocorticoids. Based on this, a safe and effective topical drug preparation that can be used to treat atopic dermatitis is urgently needed by society.

[0003] Baicalein is a monomer compound extracted and separated from the traditional Chinese medicine Scutellaria baicalensis. Its chemical name is 5,6,7-trihydroxyflavone (5,6,7-trihydroxyflavone) and its molecular formula is C 15 H 10 O5 (MW.270.24). Baicalein has multiple pharmacological effects such as antibacterial, antiviral, antiallergic, and immunomodulatory, and is one of the flavonoid compounds with the highest content in Scutellaria baicalensis.

[0004] At present, there is no patent report on the application of baicalin external preparations in the treatment of atopic dermatitis. The present invention is designed to use an external preparation dosage form to explore the application of baicalin in the treatment of atopic dermatitis. Summary of the invention

[0005] Purpose of the invention: The purpose of the present invention is to provide a baicalin external preparation and prove its good application effect in the treatment of atopic dermatitis.

[0006] The compound baicalein described in the present invention has a structural formula as shown below:

[0007]

[0008] Technical solution: The baicalin external preparation of the present invention is a liquid pharmaceutical preparation prepared by dissolving the raw material powder of baicalin or its pharmaceutically acceptable salt in a clear solution obtained by mixing any one solvent of PEG300, PEG400, propylene glycol, and ethanol or a plurality of solvents.

[0009] The baicalein external preparation and the liquid drug preparation are sprays.

[0010] The baicalein external preparation is a liquid drug preparation poured and sealed in a single open container, the liquid drug preparation is released from the container into the atmosphere through the accessories of the open container and at atmospheric pressure, and the liquid drug preparation can form liquid particles with a particle size of micrometer level after entering the atmosphere, and the liquid particles can adhere to the outer layer of keratinocytes or mucosal epithelial cells of an organism.

[0011] The baicalein external preparation, the liquid drug preparation or the ointment or gel preparation can be made by further adding pharmaceutical excipients to improve the viscosity of the preparation. Preferably, the viscosity of the preparation is further changed so that the preparation can be attached to the keratinocyte and mucosal cell layer without using a specific container and device formed by the atmospheric pressure principle.

[0012] The external preparation of baicalein, the pharmaceutical excipients include one or more of a solvent, an oil phase matrix, an emulsifier, a thickener, a pH regulator, and an antibacterial agent; wherein the solvent includes glycerol and water, the oil phase matrix includes liquid paraffin, vaseline, stearic acid, glyceryl monostearate, and white beeswax, the emulsifier includes Tween 80 and polyoxyethylene hydrogenated castor oil RH40, the thickener includes sodium carboxymethyl cellulose, xanthan gum, sodium hydroxypropyl methylcellulose, aqueous gel matrix carbomer, chitosan, sodium alginate, sodium carboxymethyl cellulose, sodium hydroxypropyl methylcellulose, the pH regulator includes triethanolamine, acetic acid, and hydrochloric acid, and the antibacterial agent includes benzalkonium chloride.

[0013] The external preparation of baicalein, the spray is a clear and transparent solution.

[0014] The external preparation of baicalein, the clear and transparent solution is a solution that is colorless, light yellow, bright yellow, or dark yellow to the naked eye.

[0015] Application of the external preparation of baicalin in atopic dermatitis medicine.

[0016] The application, the atopic dermatitis is a pathological phenomenon in which an organism is exposed to unknown substances or causes an inflammatory reaction and manifests itself on the surface of the skin or mucous membrane.

[0017] The application exerts an inhibitory effect on inflammation after contacting the keratinocytes or mucosal cell layer of an organism having atopic dermatitis.

[0018] In the external preparation of baicalein, the mass volume ratio of baicalein to other solvents or excipients is 0.005-0.015 g / mL, preferably 0.01 g / mL.

[0019] Baicalein is prepared into an external preparation that can be used for atopic dermatitis. The anti-inflammatory and antibacterial pharmacological effects of the external preparation of baicalein are beneficial to the recovery of chronic eczematous skin lesions. At the same time, the anti-allergic and immunomodulatory effects of baicalein are also beneficial to alleviate the clinical symptoms of itching of atopic dermatitis.

[0020] Beneficial effects: The topical preparation can significantly improve the pathological signs of atopic dermatitis skin tissue, reduce the transcription level of inflammatory factors in the lesion site, significantly reduce the degree of transepidermal water loss in the lesion site of atopic dermatitis skin tissue in mice, and repair the skin barrier. Its effect is better than levocetirizine, a commonly used drug for atopic dermatitis. Compared with the prior art, the topical preparation of baicalin has a more significant therapeutic effect on atopic dermatitis by the commonly used route of administration of baicalin (oral). The present invention proves through experiments that the topical preparation of baicalin has a significant effect in treating atopic dermatitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The appearance and properties of the baicalein external preparation in Example 4;

[0022] Figure 2 The results of the effect of the baicalin external preparation on the skin of atopic dermatitis-like mice in Example 5;

[0023] Figure 3 The results of the effect of the baicalin external preparation on the pathological changes of the skin tissue of atopic dermatitis-like mice in Example 5;

[0024] Figure 4 The effect of the baicalein external preparation in Example 5 on mast cell infiltration in the skin of atopic dermatitis-like mice;

[0025] Figure 5 The effects of baicalein oral administration and external application on epidermal involucrin and filaggrin in eczematoid mice in comparative example 1;

[0026] Figure 6 This is the effect of baicalein gavage and external preparations in comparative example 1 on mast cell infiltration in the skin of eczema-like mice. DETAILED DESCRIPTION

[0027] The preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are provided only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0028] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0029] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0030] Example 1

[0031] A preparation method of baicalin spray external preparation

[0032] Baicalein is highly hydrophobic and poorly water soluble. This example systematically studies the solubility of baicalein in different excipients.

[0033] Research Methodology:

[0034] Method 1: Take the raw material baicalin (batch number: HR228W3) and place it in an appropriate amount of excipients or excipient solution, and observe the dissolution phenomenon by ultrasound. After centrifugation at 10000rpm for 10 minutes, take the supernatant and filter it through a 0.45μm PES filter membrane, and take the filtrate with or without dilution to detect the content by HPLC method or UV spectrophotometry.

[0035] Method 2: Weigh the raw material baicalin (batch number: HR228W3), add it to the excipients, and observe the dissolution phenomenon by ultrasound. If it is clearly dissolved, continue to add and record the amount of raw material added.

[0036] Table 1 Saturated solubility of baicalein in different excipients or excipient solutions

[0037] medium Saturated solubility (mg / mL) Monolinoleylglycerol 0.2351 Glyceryl Monooleate 0.2148 Medium Chain Glycerides 0.0625 Labrasol 3.2761 Propylene glycol 11.67 5% Tween 80 0.9673 <![CDATA[PEG300 【1】 ]]> >50mg / mL <![CDATA[PEG400 【1】 ]]> >50mg / mL 50% ethanol 1.45mg / mL 75% ethanol 5.9 mg / mL

[0038] Note [1]: Except for this auxiliary material, which adopts method 2, all other auxiliary materials adopt method 1.

[0039] The above results (Table 1) show that baicalein has a high solubility in PEG300, PEG400, propylene glycol, and 75% ethanol. Therefore, the present invention uses PEG300, propylene glycol, and ethanol as solvents to prepare baicalein spray.

[0040] Prepared according to the following recipe process:

[0041] Table 2 Baicalein spray prescription (300mL)

[0042]

[0043] Preparation method:

[0044] 1. Measure the prescribed amount of PEG300, propylene glycol (if included in the prescription), and ethanol (if included in the prescription) and mix them evenly to obtain mixture 1;

[0045] 2. Weigh the prescribed amount of baicalin, add it to mixture 1, stir evenly, and then sonicate for 5 to 30 minutes to obtain mixture 2 after dissolution;

[0046] 3. Measure the prescribed amount of purified water, add it to mixture 2, stir evenly to obtain a yellow-brown clear solution.

[0047] Table 3 Application feeling of baicalin spray

[0048]

[0049] The above results (Table 3) show that when the amount of propylene glycol increases, the stickiness of the application increases; when the amount of ethanol increases, the application feels fresher. Considering that the basic skin symptoms of atopic dermatitis patients are dry skin and chronic eczema, attention should be paid to moisturizing properties in clinical medication. The properties of propylene glycol are similar to those of glycerin and have good moisturizing properties, so spray 1 is selected as the optimal prescription.

[0050] Example 2

[0051] Stability study of an external preparation of baicalin by atmospheric pressure device

[0052] The topical preparation was prepared according to the optimal prescription in Example 1 and placed under accelerated conditions (40°C ± 2°C, RH 75% ± 5%) and long-term conditions (25°C ± 2°C, RH 60% ± 5%). Samples were taken on 0, 5, 9 and 15 days to detect the content and related substances, wherein the statistical method for related substances was the area normalization method.

[0053] Table 4 Baicalein atmospheric pressure device spray external preparation 6 prescription (according to 300ml package)

[0054]

[0055]

[0056] Preparation process: Measure the prescribed amount of PEG300 and propylene glycol and mix them evenly to obtain mixture 1; weigh the prescribed amount of baicalein, add it to mixture 1, stir evenly and ultrasonicate for 5 to 30 minutes to dissolve it, and obtain mixture 2; measure the prescribed amount of purified water, add it to mixture 2, stir evenly to obtain a yellow-brown clear solution.

[0057] Table 5 Stability test results of baicalin spray external preparation 6 in atmospheric pressure device

[0058]

[0059] The above results (Table 5) show that the content and related substances of the topical preparation are relatively stable under accelerated conditions (40°C ± 2°C, RH 75% ± 5%) and long-term conditions (25°C ± 2°C, RH 60% ± 5%) within 15 days.

[0060] Example 3

[0061] A method for preparing an external preparation of baicalein after further adding pharmaceutical excipients

[0062] The color of baicalein raw material is dark, and the liquid particles of the preparation released by atmospheric pressure are easily stained with clothes, inconvenient to clean, and have low adhesion. Considering the compliance and convenience of clinical patients, this example attempts to further add suitable pharmaceutical excipients to the preparation, improve the viscosity of the preparation, increase the adhesion of the drug, and enable the drug to be released without atmospheric pressure device. Prepared according to the following prescription and process:

[0063] Table 6 Prescription of external preparation of baicalein after further adding pharmaceutical excipients

[0064]

[0065] Preparation method:

[0066] 1. Preparation of oil phase: Weigh the prescribed amount of oil phase, heat to 75℃ and melt for later use;

[0067] 2. Preparation of drug-containing solution: weigh the prescribed amount of API, PEG300, propylene glycol, and ethanol, mix them evenly, heat at 75°C to dissolve, and use as the drug-containing solution for later use;

[0068] 3. Preparation of aqueous phase: weigh the prescribed amount of purified water, heat it to 75°C, add sodium carboxymethyl cellulose, and after it dissolves, add polyoxyethylene hydrogenated castor oil RH40 and set aside;

[0069] 4. Preparation: Keep the temperature at 75℃, slowly add the oil phase to the water phase, high shear at 3000rpm~6000rpm, time 5~30min. After obtaining a uniform preparation, slowly add the drug-containing solution, continue high shear at 3000rpm~6000rpm, time 5~30min. Cool down to obtain.

[0070] Table 7 Appearance and properties of the preparation

[0071]

[0072] The above results (Table 7) show that formulations 2 to 4 can all be prepared with moderate viscosity after cooling and uniformity without stratification.

[0073] Example 4

[0074] Method 2 for preparing an external preparation of baicalein cream after further adding pharmaceutical excipients

[0075] In order to increase the advantages of the preparation such as easy spread, comfortable feeling, non-greasy, easy to wash off, and not interfering with the normal physiological function of the skin, based on this, this embodiment provides a preparation method of a new preparation to explore its feasibility.

[0076] Prepared according to the following prescription and process:

[0077] Table 8 Prescription of external preparation of baicalein after further adding pharmaceutical excipients

[0078]

[0079] Preparation method:

[0080] 1. Weigh the prescribed amount of carbomer and add it to the prescribed amount of water. Leave it overnight until it swells naturally. Then add the prescribed amount of triethanolamine while stirring until a transparent colloidal liquid is formed.

[0081] 2. Preparation of drug-containing solution: Weigh the prescribed amount of baicalein, PEG300, propylene glycol (if included in the prescription), glycerol (if included in the prescription), and ethanol (if included in the prescription), stir evenly, and sonicate for 5 to 30 minutes. After dissolving, add the prescribed amount of 50% benzalkonium chloride ethanol solution, mix evenly, and set aside;

[0082] 3. Add the drug-containing solution to the colloidal liquid in "Step 1", stirring while adding, mix evenly to form a yellow transparent gel.

[0083] Table 9: Application feeling of the preparations

[0084]

[0085] Comparison of the appearance of different formulations of the drug ( Figure 1 ), application feeling (Table 9), the results show that Preparation 8 can obtain better appearance and more comfortable application feeling.

[0086] Example 5

[0087] Study on the main pharmacodynamic effects of baicalin topical preparations in the treatment of atopic dermatitis

[0088] 1. Experimental Materials

[0089] (1) Drugs

[0090] Example 1 Baicalein external preparation (10 mg / ml)

[0091] (2) Experimental animals

[0092] C57BL / 6J mice, female, 8 weeks old, were housed in an SPF environment with a temperature of 20-25°C, a relative humidity of 30-70%, and a 12:12h light-dark lighting schedule; they had free access to water and food.

[0093] (3) Reagents

[0094] 1) Calcipotriol (MC903): MCE Company, product number: HY-10001.

[0095] 2) Positive drug: Levocetirizine: MCE Company, product number: HY-B0814.

[0096] 3) PBS solution: NaCl 8.0 g, KH2PO4 2.0 g, Na2HPO4H2O 1.56 g, KCl 0.20 g, dissolved in 1000 mL ultrapure water, sterilized by high pressure steam, and stored at 4°C.

[0097] 2. Experimental methods:

[0098] 19 model mice were randomly divided into 5 groups according to body weight, namely blank control group, model + solvent control group, positive drug control group, baicalin low-dose group and baicalin high-dose group, with 6 mice in each group. The grouping is shown in the table below:

[0099] Table 10 Animal grouping

[0100]

[0101] Except for the blank group, 10 nmol of MC903 was applied to the left ear skin of mice in other groups to establish a mouse atopic dermatitis model. After modeling, different doses of the test samples were given for treatment according to the grouping. The test sample group was administered by applying the affected skin of the mice, once a day, for 13 consecutive days, and the positive drug control group was gavage, once a day, for 13 consecutive days, and the administration volume was 0.1 mL / 10 g. The ear edge thickness of the mice was tested every three days for seven days before the experiment, and every two days for seven days after the experiment. On the last day of the experiment, the mice were killed by cervical dislocation, and the ear tissue of the same area was taken to weigh its weight.

[0102] HE staining was used to analyze the pathological changes of mouse ear tissues, and the epidermal thickness was calculated and statistically analyzed.

[0103] The transcription levels of IL-4 and TSLP in the mouse model skin tissue were detected by real-time fluorescence quantitative PCR.

[0104] 3. Statistical methods:

[0105] All measurement data are The comparison among multiple groups was performed by one-way analysis of variance (ANOVA). P < 0.05 was considered statistically significant.

[0106] 4. Experimental results:

[0107] Table 11 Effect of external application of baicalin on the thickness of the ear margin of mice ( n=6)

[0108]

[0109] #P<0.05, ####P<0.0001 compared with the blank control group, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 compared with the model+solvent control group.

[0110] Table 12 Effect of external application of baicalin on ear margin thickness of mice ( n=6)

[0111]

[0112] #P<0.05, ####P<0.0001 compared with the blank control group, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 compared with the model+solvent control group.

[0113] Table 13 Effect of topical baicalin on epidermal thickness in atopic dermatitis-like mice

[0114]

[0115] #P<0.05, ####P<0.0001 compared with the blank control group, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 compared with the model+solvent control group.

[0116] Table 14 Effect of topical baicalein on the expression of specific cytokines in atopic dermatitis mice ( n=6)

[0117]

[0118] #P<0.05, ####P<0.0001 compared with the blank control group, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 compared with the model+solvent control group.

[0119] Table 15 Effect of topical baicalein on mast cell infiltration in the skin of atopic dermatitis-like mice

[0120]

[0121] #P<0.05, ####P<0.0001 compared with the blank control group, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 compared with the model+solvent control group.

[0122] Effect of topical baicalin on MC903-induced atopic dermatitis-like symptoms ( Figure 2 ) showed that topical baicalin can significantly alleviate atopic dermatitis-like symptoms.

[0123] The measurement results of the ear margin thickness of mice after external application of baicalin (Table 11 and Table 12) showed that the ear margin thickness of mice in the control group fluctuated within a certain range during the experiment and did not increase significantly; the ear margin thickness of mice in the model group increased significantly with the passage of modeling time; the external application of baicalin inhibited the increase of the ear margin thickness of mice to a certain extent, and the effect was equivalent to that of the positive drug.

[0124] Effect of external application of baicalin on pathological changes of mouse ear tissues ( Figure 3 and Table 13) showed that topical baicalein could significantly inhibit the thickening of the mouse epidermis.

[0125] The effects of topical baicalin on the transcriptional levels of IL-4 and TSLP (Table 14) showed that the transcriptional levels of IL-4 and TSLP in the model group were significantly increased compared with the blank control group. In the group treated with topical baicalin, baicalin significantly reduced the transcriptional levels of IL-4 and TSLP, achieving the effect of reducing the level of specific cytokines and improving the symptoms of atopic dermatitis.

[0126] Effect of topical baicalin on mast cell infiltration in the skin of atopic dermatitis-like mice (Table 15, Figure 4 ) showed that compared with the blank control group, the level of mast cells in the model group was significantly increased. In the group treated with external baicalin, baicalin significantly reduced the level of mast cells, and the effect was equivalent to that of the positive drug.

[0127] The above results show that baicalin topical preparations can play a role in the treatment of atopic dermatitis.

[0128] Comparative Example 1

[0129] Comparative test of the commonly used oral administration route of baicalin and the baicalin external preparation of the present invention (Example 1). The experimental results of animals showed that both oral administration and external administration of baicalin had significant inhibitory effects on atopic dermatitis, but the therapeutic effect of baicalin external preparation was better, including its inhibitory effect on mouse skin thickness (Table 16), its effect on mouse epidermal involucrin and filaggrin ( Figure 5 ) and its inhibitory ability on mast cell infiltration in mouse skin ( Figure 6) are superior to the commonly used oral administration route of baicalein.

[0130] Table 16 Comparison of the efficacy of baicalin administered orally and skin preparations in AD-like mouse model

[0131]

[0132]

[0133] #P<0.05, ####P<0.0001 compared with blank control group, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 compared with model control group.

[0134] Comparative Example 2

[0135] The disclosed technology has disclosed the application of a baicalein external preparation (hereinafter referred to as "preparation BE") in atopic dermatitis [Therapeutic effects of Baicalein on atopic dermatitis-like skin lesions of NC / Nga mice induced by dermatophagoides pteronyssinus, Int Immunopharmacol. 2010 Sep; 10(9): 1142-8. doi: 10.1016 / j.intimp.2010.06.020. Epub 2010 Jul 9.]. The technical data results show that the reduction level of IL-4, an inflammatory index in atopic dermatitis, by preparation BE is 31.16%, which is lower than the 92.61% reduction level achieved by the present technology. This shows that the therapeutic effect of the baicalein external preparation prepared by the present technology is more significantly superior than that of the prior art.

Claims

1. A baicalein external preparation, It is characterized in that The external preparation is a spray, and the prescription is 300 mL: 。 2. The external preparation of baicalin according to claim 1, It is characterized in that The topical preparation is poured and sealed in a single open container, and the topical preparation is released from the container into the atmosphere through the accessories of the open container in an atmospheric pressure manner. After entering the atmosphere, the topical preparation can form liquid particles with a particle size of micrometer level, and the liquid particles can adhere to the outer layer of keratinocytes or mucosal epithelial cells of the organism.

3. The external preparation of baicalein according to claim 1, It is characterized in that The external preparation can be made into an ointment or a gel after further adding pharmaceutical excipients to improve the viscosity of the preparation.

4. The external preparation of baicalin according to claim 3, It is characterized in that The pharmaceutical excipients include one or more of a solvent, an oil phase matrix, an emulsifier, a thickener, a pH regulator, and an antibacterial agent; wherein the solvent includes glycerol and water, the oil phase matrix includes liquid paraffin, vaseline, stearic acid, glyceryl monostearate, and white beeswax, the emulsifier includes Tween 80 and polyoxyethylene hydrogenated castor oil RH40, the thickener includes sodium carboxymethyl cellulose, xanthan gum, sodium hydroxypropyl methylcellulose, an aqueous gel matrix carbomer, chitosan, and sodium alginate, the pH regulator includes triethanolamine, acetic acid, and hydrochloric acid, and the antibacterial agent includes benzalkonium chloride.

5. The external preparation of baicalin according to claim 1, It is characterized in that The spray is a clear and transparent solution.

6. The external preparation of baicalin according to claim 5, It is characterized in that The clear and transparent solution is a solution that is colorless, light yellow, bright yellow or dark yellow to the naked eye.

7. Use of the external preparation of baicalin according to any one of claims 1 to 6 in the preparation of atopic dermatitis drugs.

8. The use according to claim 7, It is characterized in that Atopic dermatitis is a pathological phenomenon in which an organism is exposed to unknown substances or causes an inflammatory reaction and manifests itself on the surface of the skin or mucous membranes.

9. The use according to claim 7, It is characterized in that It exerts an inhibitory effect on inflammation after contact with the keratinocytes or mucosal cell layer of organisms with atopic dermatitis.

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