A pharmaceutical composition for treating chapped skin caused by tinea pedis (athlete's foot).

Abstract

This invention discloses a pharmaceutical composition for treating chapped skin caused by tinea pedis and manuum. The composition comprises urea, olive leaf extract, oat alkaloids, and asiaticoside as active ingredients, combined with a hypoallergenic liquid crystal emulsion matrix consisting of shea butter, squalane, caprylic / capric triglycerides, and cetearyl glucoside. This invention utilizes urea to soften keratin and promote crack healing, while simultaneously inhibiting the irritation caused by urea through the synergistic effect of three plant-based anti-inflammatory components, and combining biomimetic oils to repair the skin's lipid barrier. This composition significantly reduces pain and itching at the chapped areas in patients with tinea pedis and manuum, promotes crack closure, and avoids the burning and irritation commonly found in traditional antifungal ointments.

Description

Technical Field

[0001] This invention belongs to the field of medicine and relates to a low-irritant pharmaceutical composition for treating chapped skin caused by tinea pedis and its preparation method and application. Background Technology

[0002] Tinea pedis and manuum are common superficial fungal infections of the palms, soles, and interdigital spaces caused by dermatophytes (mainly Trichophyton rubrum and Trichophyton mentagrophytes). Chronic tinea pedis and manuum patients experience hyperkeratosis, thickening, and desquamation of the stratum corneum due to long-term irritation from fungal metabolic products, eventually leading to fissures extending into the dermis. These fissures are painful and may even bleed, severely affecting the patient's walking or hand movement.

[0003] Urea is a recognized active ingredient in topical dermatological medications. As a direct supplement to natural moisturizing factor (NMF), urea significantly increases the moisture content of the stratum corneum. Simultaneously, its mild keratinolytic effect softens and removes excessively keratinized scales, thus it is widely used to treat dry skin conditions such as chapped hands and feet, ichthyosis, and chronic eczema. Currently, 10% urea ointment is a commonly used over-the-counter topical preparation in clinical practice.

[0004] However, the clinical treatment of chapped skin caused by tinea pedis and manuum faces a dilemma: conventional antifungal drugs (such as terbinafine and miconazole) are effective against fungi, but their alcohol-based bases or irritating excipients can cause severe burning and stinging when applied to chapped wounds, leading to poor patient compliance. While using 10% urea ointment alone can soften keratin and promote wound healing, high concentrations of urea also cause significant stinging, burning, and erythema, especially in chapped areas where the skin barrier is already damaged. Furthermore, traditional urea ointments often use lanolin, mineral oil, and PEG-based emulsifiers as their base. While lanolin and mineral oil have good occlusive properties, some people are allergic to them, and they are greasy and difficult to wash off; PEG-based emulsifiers may damage the already compromised skin barrier, increasing the risk of transdermal absorption and further inducing irritation.

[0005] In existing technologies, corticosteroids, antihistamines, or traditional anti-inflammatory plant extracts are often added to alleviate urea irritation. However, corticosteroid preparations carry risks such as skin atrophy and steroid-dependent dermatitis; single anti-inflammatory components often have limited efficacy and poor compatibility with urea at high concentrations. Therefore, developing a pharmaceutical composition that can maintain the efficacy of 10% urea in softening keratin and promoting crack healing, while significantly reducing its irritation, and with an overall mild, skin-friendly, and safe matrix, is of significant clinical importance for the treatment of fissures caused by tinea pedis and manuum. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a low-irritation, highly effective pharmaceutical composition for repairing chapped skin caused by tinea pedis and tinea manuum. This composition achieves optimal therapeutic effects by combining moisturizing and repairing active ingredients with soothing and anti-inflammatory mechanisms, along with a low-allergenic oil system. It significantly improves skin tolerance while maintaining the optimal efficacy of 10% urea, making it specifically suitable for adjunctive treatment of chapped skin in patients with tinea pedis and tinea manuum.

[0007] In a first aspect, the present invention provides a pharmaceutical composition for treating chapped skin caused by tinea pedis, comprising the following components in weight percentages: urea 8%~12%; olive leaf extract 0.1%~3%; oat alkaloids 0.01%~0.5%; asiaticoside 0.05%~1%; shea butter 1%~8%; squalane 2%~10%; caprylic / capric triglycerides 3%~12%; cetearyl glucoside 0.5%~4%; and a pharmaceutically acceptable carrier.

[0008] Preferably, the specific point values ​​for each of the above components within the stated range can be selected as needed. Specifically: The amount of urea used is selected from 8%, 9%, 10%, 11% or 12%, preferably 10%.

[0009] The amount of the olive leaf extract is selected from 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5% or 3%, preferably 0.5% to 2%, for example 1%.

[0010] The amount of oat alkaloids used is selected from 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4% or 0.5%, preferably 0.05% to 0.2%, for example 0.1%.

[0011] The amount of asiaticoside used is selected from 0.05%, 0.1%, 0.2%, 0.3%, 0.5%, 0.8% or 1%, preferably 0.1% to 0.5%, for example 0.2%.

[0012] The amount of shea butter used is selected from 1%, 2%, 3%, 4%, 5%, 6%, 7% or 8%, preferably 3% to 6%, for example 5%.

[0013] The amount of squalane used is selected from 2%, 4%, 6%, 8% or 10%, preferably 4% to 8%, for example 6%.

[0014] The amount of caprylic / capric triglyceride is selected from 3%, 5%, 7%, 9% or 12%, preferably 5% to 10%, for example 8%.

[0015] The amount of cetearyl glucoside used is selected from 0.5%, 1%, 2%, 3% or 4%, preferably 1% to 3%, for example 2%.

[0016] In this composition, urea serves as the core moisturizing and keratin repair agent, providing keratin softening and moisture replenishment, softening the thickened palmar and plantar keratin in patients with tinea pedis and promoting the shedding of necrotic tissue at the edges of fissures; olive leaf extract, rich in oleuropein, can inhibit COX-2 and inducible nitric oxide synthase, reducing the production of inflammatory mediators, and has also been reported in the literature to have a certain inhibitory effect on dermatophytes; oat alkaloids are natural anti-inflammatory and antipruritic components that can antagonize substance P and inhibit the NF-κB pathway, effectively relieving itching and neurogenic inflammation associated with fissures; asiaticoside, the main active ingredient of Centella asiatica, can promote collagen synthesis, inhibit mast cell degranulation, and accelerate the filling of granulation tissue in fissures. These four active ingredients work synergistically through different pathways, significantly reducing the irritation to damaged skin while ensuring the efficacy of urea.

[0017] Regarding the oil matrix, this invention abandons lanolin, mineral oil, and irritating emulsifiers found in traditional formulations, opting instead for shea butter, rich in unsaturated fatty acids and terpenes, which possesses repairing and anti-inflammatory properties. It selects squalane and caprylic / capric triglycerides, natural components of the sebum film with excellent skin affinity, as the oil phase matrix, and combines them with cetearyl glucoside (a non-ionic emulsifier that can form a liquid crystal structure). This oil system exhibits good skin affinity and low sensitization, helping to repair the sebum film surrounding cracks and further enhancing the formula's gentleness.

[0018] Furthermore, the pharmaceutically acceptable carriers include humectants, preservatives, pH adjusters, and solvents.

[0019] Preferably, the humectant may be selected from one or more of glycerin, propylene glycol, butylene glycol, and sorbitol, and the amount used is 1% to 10%; the preservative may be selected from one or more of phenoxyethanol, p-hydroxyacetophenone, and ethylhexylglycerin, and the amount used is 0.1% to 1.0%; the pH adjuster may be selected from one or more of triethanolamine, citric acid, and sodium citrate; and the solvent may be water, preferably deionized water. The pH of the composition is adjusted to 5.5 to 6.5.

[0020] As a preferred embodiment, the pharmaceutical composition further comprises a co-emulsifier, cetearyl alcohol, at a content of 1% to 4%, for stabilizing the liquid crystal emulsion structure. The pharmaceutical composition is free of lanolin, mineral oil, and PEG-based emulsifiers.

[0021] In a second aspect, the present invention provides a method for preparing a pharmaceutical composition, comprising the following steps: (1) Preparation of oil phase: Shea butter, squalane, caprylic / capric triglyceride, cetearyl glucoside, and optional co-emulsifier cetearyl alcohol are weighed and mixed according to the formula, heated to 75-80℃, stirred until completely melted into a transparent and uniform liquid, and kept warm for later use.

[0022] (2) Aqueous phase preparation: Weigh and mix water-soluble excipients such as deionized water, urea, humectant, and preservative according to the ratio, heat to 75-80℃, stir until completely dissolved, and keep warm for later use.

[0023] (3) Emulsification: Under homogenization and stirring conditions, the aqueous phase is slowly added to the oil phase. After the addition is complete, homogenization continues for 3-5 minutes to form a liquid crystal emulsion. The homogenization speed is 5000-12000 rpm, preferably 6000-8000 rpm.

[0024] (4) Cooling and adding heat-sensitive active ingredients: Turn on the jacket cooling water to cool the emulsion system to below 45°C, preferably to 40-45°C. Add olive leaf extract, oat alkaloids and asiaticoside, and stir slowly (300-500 rpm) to disperse them evenly.

[0025] (5) pH adjustment and finished product: Adjust the pH of the system to 5.5-6.5 with triethanolamine. Continue stirring and cool to room temperature, let stand to remove bubbles, and the cream-like drug composition is obtained.

[0026] Secondly, the present invention also provides a method for preparing the olive leaf extract in the pharmaceutical composition, comprising the following steps: (1) Extraction: Take dried olive leaves, remove impurities, and pulverize them through a 20-40 mesh sieve to obtain olive leaf powder. Add 6-12 times (volume / weight ratio) of 50%-80% ethanol to the olive leaf powder, and heat and reflux in a water bath at 50-80℃ for 1-3 times, each time for 1-3 hours. Filter and combine the filtrates.

[0027] Preferably, the amount of ethanol added is 8-10 times the volume; the volume fraction of ethanol is 60%-70%; the extraction temperature is 60-70℃; the extraction is performed twice; and the extraction time for each extraction is 1-2 hours.

[0028] (2) Concentration: The filtrate is concentrated under reduced pressure at 40-60℃ to an extract with a relative density of 1.05-1.20 (measured at 60℃).

[0029] Preferably, the concentration temperature is below 50°C; and the concentration is carried out to a relative density of 1.10-1.15.

[0030] (3) Purification: Disperse the extract with an appropriate amount of water and load it onto a macroporous adsorption resin column (such as D101 or AB-8). First, elute with 2-3 column volumes of deionized water to remove polar impurities such as sugars, and then elute with 3-5 column volumes of ethanol with a volume fraction of 20%-60%. Collect the ethanol eluent.

[0031] Preferably, the volume fraction of ethanol elution is 30%-50%.

[0032] (4) Drying: The ethanol eluent is concentrated under reduced pressure below 50°C, vacuum dried, or freeze-dried to obtain olive leaf extract powder. The content of oleuropein in the obtained extract is ≥20%.

[0033] Thirdly, the pharmaceutical composition provided by the present invention can be used to prepare a drug for treating chapped skin caused by tinea pedis.

[0034] Those skilled in the art know that in patients with chapped skin caused by tinea pedis, the skin on the palms and soles is excessively thickened and cracked due to long-term fungal infection, with cracks extending deep into the dermis, accompanied by significant pain, bleeding, and itching. The irritating base of conventional antifungal ointments can cause a burning sensation, while simple urea ointment also presents a stinging problem.

[0035] In the pharmaceutical composition of the present invention, urea softens the thickened stratum corneum, peels away necrotic tissue, and increases local moisture content, creating conditions for crack healing; olive leaf extract, oat alkaloids, and asiaticoside synergistically inhibit the inflammatory response, reduce the stinging and burning sensation caused by urea, and at the same time promote fibroblast proliferation and collagen synthesis, accelerating crack healing; shea butter, squalane, caprylic / capric triglycerides, and liquid crystal emulsion matrix provide a biomimetic lipid environment, repair the sebum film, and reduce transepidermal water loss.

[0036] The medicine of this invention, in addition to antifungal treatment (such as topical terbinafine or oral antifungal drugs) for patients with tinea pedis and manuum, involves applying an appropriate amount of the composition of this invention to the cracked area and surrounding skin twice daily for 1-2 weeks. This composition serves as an adjunctive treatment specifically for addressing cracking issues accompanying tinea pedis and manuum, without interfering with the efficacy of antifungal drugs.

[0037] Compared with the prior art, the present invention has the following significant advantages: 1. The present invention specifically addresses the clinical contradiction between the strong irritation of antifungal drugs and the strong irritation of simple urea ointment. Through the synergistic effect of three natural anti-inflammatory components, it completely eliminates or significantly reduces the stinging and burning sensation of 10% urea on cracked wounds.

[0038] 2. Urea softens the stratum corneum and replenishes NMF, while squalane and shea butter directly replenish the sebum film components, and asiaticoside promotes collagen synthesis, achieving a good repair effect.

[0039] 3. The formulation of this application eliminates known allergens (lanolin, mineral oil, PEG emulsifiers) and uses liquid crystal emulsification technology to simulate the natural lipid bilayer structure of the skin, making it suitable for use on broken and cracked areas. Detailed Implementation

[0040] The present invention will be further illustrated below through specific embodiments. It should be understood that these embodiments are for illustrative purposes only and do not constitute a limitation on the scope of protection of the present invention.

[0041] Example 1 Matrix screening experiment formulation design To verify the superiority of the oil matrix and emulsification system described in this invention, the following blank matrix formulations (all of which do not contain urea and three plant anti-inflammatory components) were set up to compare the effects of differences in oil / emulsifier composition on matrix performance.

[0042] 1. Blank matrix formulation composition The total amount of each blank matrix was 100g, with any shortfall made up with deionized water. It was divided into: (1) Blank matrix A (mineral oil + lanolin system): Liquid paraffin 8.0g, lanolin 5.0g, caprylic / capric triglyceride 8.0g, cetearyl alcohol 2.0g, glycerin 5.0g, phenoxyethanol 0.5g, deionized water to 100g.

[0043] Blank matrix B (PEG emulsion system): Shea butter 5.0g, squalane 6.0g, caprylic / capric triglyceride 8.0g, PEG-100 stearate 2.0g, glyceryl monostearate 2.0g, cetearyl alcohol 2.0g, glycerin 5.0g, phenoxyethanol 0.5g, deionized water to 100g.

[0044] Blank matrix C (using squalane alone, lacking shea butter): Squalane 11.0g, Caprylic / Capric Triglyceride 8.0g, Cetearyl Glucoside 2.0g, Cetearyl Alcohol 2.0g, Glycerin 5.0g, Phenoxyethanol 0.5g, Deionized Water to 100g.

[0045] Experimental matrix (i.e., the liquid crystal emulsion matrix, which does not contain active ingredients): Shea butter 5.0g, squalane 6.0g, caprylic / capric triglyceride 8.0g, cetearyl glucoside 2.0g, cetearyl alcohol 2.0g, glycerin 5.0g, phenoxyethanol 0.5g, deionized water to 100g.

[0046] 2. Preparation method Each of the above blank matrices was prepared according to the following steps: (1) Preparation of oil phase: Mix the oil components (shea butter, squalane, caprylic / capric triglyceride, liquid paraffin, lanolin, etc., according to their respective formulas), emulsifiers (cetearyl glucoside, PEG-100 stearate, glyceryl monostearate, etc., according to their respective formulas) and cetearyl alcohol, heat to 75-80℃, stir until completely melted into a uniform liquid, and keep warm for later use.

[0047] (2) Aqueous phase preparation: Mix deionized water, glycerol and phenoxyethanol, heat to 75-80℃, stir until completely dissolved, and keep warm for later use.

[0048] (3) Emulsification: Under the condition of keeping warm at 75-80℃, turn on the homogenizer (7000rpm) and slowly add the aqueous phase to the oil phase. The feeding time is about 1-2 minutes. After the feeding is completed, continue homogenization for 4 minutes.

[0049] (4) Cooling: Turn on the cooling to cool the emulsion system to below 45°C and continue stirring for 15 minutes.

[0050] (5) pH adjustment: Adjust the pH to 5.5-6.5 with 10% triethanolamine, continue stirring and cool to room temperature, let stand to remove bubbles, and obtain blank matrix.

[0051] Example 2 Test method: 1. Subjects and Testing Environment Thirty healthy volunteers, aged 18-60, with no history of skin diseases, and who have not used any topical products on the test site within 7 days prior to the test, will be recruited. The test will be conducted in a temperature and humidity controlled laboratory, with the temperature controlled at 21±1℃ and the relative humidity controlled at 50±5%. Participants are required to sit quietly in the laboratory for 30 minutes before the test to allow for acclimatization.

[0052] 2. Transdermal Water Loss (TEWL) Test Measurements were performed using a Tewameter TM300 probe. Four test zones were marked on the inner forearm of the subject (one zone for each blank matrix, plus a blank control zone without any sample). Zones were randomly assigned. A 2 mg / cm² sample was applied evenly using a finger cot. TEWL values ​​were measured before application (baseline) and 4 hours after application. Measurements were taken three times for each zone at each time point, and the average value was recorded. The TEWL change rate was calculated using the following formula: TEWL change rate (%) = (TEWL value after application - Baseline TEWL value) / Baseline TEWL value × 100% A negative TEWL change rate indicates improved barrier function and reduced water loss.

[0053] 3. Skin stratum corneum moisture content test Measurements were performed using a Corneometer CM825 probe. Measurements were taken at the same test area and time points (before application and 4 hours after application), with three consecutive measurements taken for each area at each time point, and the average value was recorded. The moisture content growth rate was calculated using the following formula: Moisture content growth rate (%) = (Moisture content after application - Baseline moisture content) / Baseline moisture content × 100% 4. Subjective greasiness rating Immediately after applying the sample, participants subjectively rated its oiliness. Rating criteria: 1 point = very refreshing, 2 points = somewhat refreshing, 3 points = moderate, 4 points = somewhat oily, 5 points = very oily. The average rating of 20 participants was used.

[0054] 5. Experimental Results The shea butter, squalane, caprylic / capric triglycerides, and cetearyl glucoside liquid crystal emulsion system of this invention (i.e., the blank matrix of this invention), without any active ingredients, exhibits significantly better barrier repair ability (TEWL decrease of 26.3%), moisturizing effect (water content increase of 50.6%), and refreshing skin feel (oiliness score of 2.0) than the traditional mineral oil / lanolin matrix (blank matrix A) and PEG emulsion system (blank matrix B). The moisturizing and barrier repair effects of blank matrix C (lacking shea butter) are significantly lower than those of the blank matrix of this invention, indicating that shea butter provides excellent and indispensable repair for the skin's lipid barrier.

[0055] Example 3 The pharmaceutical composition of this embodiment, by weight percentage, consists of the following components: 10.0g urea, 1.0g olive leaf extract, 0.1g oat alkaloids, 0.2g asiaticoside, 5.0g shea butter, 6.0g squalane, 8.0g caprylic / capric triglyceride, 2.0g cetearyl glucoside, 2.0g cetearyl alcohol, 5.0g glycerin, 0.5g phenoxyethanol, deionized water to 100g, and an appropriate amount of triethanolamine to adjust the pH to 5.5-6.5.

[0056] Olive leaf extract was prepared in advance as follows: dried olive leaves were pulverized and passed through a 30-mesh sieve. Ten times the volume of 70% ethanol was added, and the mixture was refluxed twice at 65°C for 1.5 hours each time. The extracts were filtered, and the filtrates were combined. The filtrate was concentrated under reduced pressure below 50°C to a relative density of 1.10 (measured at 60°C). The extract was dispersed in an appropriate amount of water and loaded onto a D101 macroporous adsorption resin column. The column was eluted with two column volumes of deionized water, followed by four column volumes of 40% ethanol. The ethanol eluent was collected. The ethanol eluent was concentrated under reduced pressure below 50°C and vacuum dried to obtain olive leaf extract powder. HPLC analysis showed that the content of oleuropein in the extract was 24.3%.

[0057] The composition of Example 3 was prepared using liquid crystal emulsification technology, and the preparation method includes the following steps: (1) Preparation of oil phase: Shea butter, squalane, caprylic / capric triglyceride, cetearyl glucoside and cetearyl alcohol are weighed in sequence and added to a beaker. The mixture is heated in a water bath to 75-80℃ and stirred at 300-500 rpm until all components are completely melted into a transparent and uniform liquid. The mixture is kept warm for later use.

[0058] (2) Preparation of aqueous phase: Deionized water, urea, glycerol and phenoxyethanol are added to another beaker in sequence, stirred and mixed, heated in a water bath to 75-80℃, stirred until completely dissolved, the solution is clear and transparent, and kept warm for later use.

[0059] (3) Emulsification: Under the condition of keeping warm at 75-80℃, turn on the homogenizer (speed 7000rpm) and slowly add the aqueous phase to the oil phase in a thin stream for about 1-2 minutes. After the addition is completed, continue to homogenize at 7000rpm for 4 minutes to obtain a uniform and delicate milky white liquid crystal emulsion.

[0060] (4) Cooling and adding heat-sensitive active ingredients: Turn on the cooling water circulation to quickly cool the emulsion system to below 45°C. Under slow stirring (400 rpm), add olive leaf extract, oat alkaloids and asiaticoside in sequence, and continue stirring for 15 minutes until evenly dispersed.

[0061] (5) pH adjustment and finished product: Slowly add the pre-prepared 10% triethanolamine solution dropwise to the emulsion while stirring. Monitor the pH with a pH meter and adjust it to 5.5-6.5. Continue stirring and cool to room temperature. Let it stand for 30 minutes to remove bubbles, and the cream-like drug composition is obtained.

[0062] The pharmaceutical composition prepared in Example 3 was a uniform cream ranging from off-white to light yellow in color, with a fine texture, easy application, and no grainy feel, and a pH of 6.0 ± 0.2. After being placed at 4°C, 25°C, and 40°C for 3 months, no layering, emulsification, or color change was observed, indicating good stability.

[0063] Comparative Examples 1-3 To verify the effects of the combination of urea, olive leaf extract, oat alkaloids, and asiaticoside in this invention, the following formula was prepared, with a total amount of 100g for each ingredient.

[0064] Table 1. Formulations for Comparative Examples 1-3 (Unit: g) Comparative Examples 1-3 were prepared using a method similar to that of Example 3. Similarly, the pharmaceutical compositions obtained in Comparative Examples 1-3 were also uniform creams ranging from off-white to light yellow, with a fine texture, easy application, and no grainy feel.

[0065] Example 4 In vitro anti-inflammatory synergistic evaluation (LPS-induced NO inhibition assay in RAW264.7 macrophages) 1. Experimental Materials Cell line: mouse mononuclear macrophage leukemia cell line RAW264.7.

[0066] Main reagents: LPS (lipopolysaccharide, Sigma-Aldrich), Griess reagent, DMEM high glucose medium, fetal bovine serum and CCK-8 kit.

[0067] Test samples: Samples prepared in Example 3 and samples from Comparative Examples 1-3 (formulas are shown in Table 1). Each sample was dissolved in DMSO and then diluted with DMEM medium to the required concentration, with the final DMSO concentration not exceeding 0.1%.

[0068] Positive control: Dexamethasone, diluted in DMEM medium.

[0069] 2. Cell Culture RAW264.7 cells were seeded in DMEM medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin, and routinely passaged in a 37°C, 5% CO2 incubator. Cells in the logarithmic growth phase were used in the experiments.

[0070] 3. Cytotoxicity assay (to determine safe concentration) RAW264.7 cells were fed at a concentration of 1×10⁻⁶. 4Cells were seeded per well in 96-well plates and cultured overnight. The original culture medium was discarded, and fresh culture medium containing different concentrations of the test samples (0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.2%, 0.5%, w / v) was added, with three replicates for each concentration. A blank control (without sample) and a background control (cell-free) were also included. After 24 hours of culture, 10 μL of CCK-8 reagent was added to each well, and incubation continued for 2 hours. The absorbance at 450 nm was measured using a microplate reader. Cell viability was calculated, and the highest concentration with ≥90% cell viability was selected as the non-cytotoxic concentration for subsequent experiments. Results showed that the cell viability of all test samples was greater than 90% at a concentration of 0.1% (w / v), therefore, 0.1% was used as the treatment concentration in subsequent experiments.

[0071] 4. RAW264.7 cells were cultured at a rate of 2 × 10⁻⁶. 5 Seeds were inoculated per well in a 24-well plate and incubated overnight. The experiment included the following groups: Blank control group: containing only culture medium, without cells.

[0072] Model control group: cells + LPS (1 μg / mL), without the test sample.

[0073] Example 3 group: cells + LPS (1 μg / mL) + sample of the present invention (0.1%).

[0074] Comparative Example 1: Cells + LPS (1 μg / mL) + Comparative Example 1 sample (0.1%).

[0075] Comparative Example 2: Cells + LPS (1 μg / mL) + Comparative Example 2 sample (0.1%).

[0076] Comparative Example 3: Cells + LPS (1 μg / mL) + Comparative Example 3 sample (0.1%).

[0077] Positive control group: cells + LPS (1 μg / mL) + dexamethasone (10 μmol / L).

[0078] Each group had three replicates. After drug administration, the cells were incubated at 37°C in a 5% CO2 incubator for 24 hours. The cell culture supernatant was collected, and the NO content was determined using the Griess method: 50 μL of the supernatant was added to a 96-well plate, followed by 50 μL each of Griess reagent A and B, and incubated at room temperature in the dark for 10 minutes. The absorbance at 540 nm was measured using a microplate reader. The NO concentration was calculated based on the sodium nitrite standard curve.

[0079] The NO inhibition rate is calculated using the following formula: Inhibition rate (%) = (NO concentration in the model control group - NO concentration in the drug treatment group) / (NO concentration in the model control group - NO concentration in the blank control group) × 100% 5. Results Table 2. In vitro anti-inflammatory results (NO inhibition rate) The experimental results are shown in Table 2. Compared with the model control group, all drug-treated groups showed a significant reduction in NO concentration (p<0.01). The NO inhibition rate of the Example 3 group was 78.4%, significantly higher than that of Comparative Example 1 (45.3%), Comparative Example 2 (62.1%), and Comparative Example 3 (58.7%) (p<0.01). The NO inhibition rate of the present invention group was significantly higher than that of Comparative Examples 1-3 (p<0.01).

[0080] Example 5 Evaluation of irritation relief (chicken embryo chorioallantoic membrane test, HET-CAM) 1. Experimental Materials Fertilized eggs: SPF grade white Leghorn eggs, incubated for 9-11 days.

[0081] Test samples: the sample of the present invention prepared in Example 3, the sample of Comparative Example 1, the sample of Comparative Example 2, the sample of Comparative Example 3, commercially available 10% urea ointment (positive control), and blank matrix (negative control).

[0082] Main instruments: egg candling machine, constant temperature incubator, stereo microscope, timer.

[0083] 2. Experimental Procedure The HET-CAM method was followed according to OECD Guideline TG 442, with appropriate adjustments for testing paste-like samples. Chicken embryos on day 10 of incubation were used; the air cell portion of the shell and shell membrane was removed to expose the chorioallantoic membrane (CAM). 0.2g of the test sample was evenly applied to the CAM surface. Six chicken embryos were tested per group.

[0084] Observation and Recording: Within 5 minutes of contact with the sample, record the time (in seconds) at which CAM bleeding, angiolysis, or clotting occurs. If no phenomenon occurs within 300 seconds, record that time as 301 seconds.

[0085] 3. Stimulus Score Calculation The stimulus score (IS) is calculated using the following formula: IS =[(301- tH )+(301- tL )+(301- tC )] / 30 in , , These represent the time (in seconds) for bleeding, angiolysis, and clotting, respectively. A smaller IS value indicates weaker irritation. Judgment criteria: IS < 3 indicates no irritation, 3 ≤ IS < 6 indicates mild irritation, and IS ≥ 6 indicates moderate or stronger irritation.

[0086] 4. Results The experimental results are shown in Table 3.

[0087] Table 3 Evaluation results of irritation relief The IS score of Example 3 group was 1.2, lower than the mild irritation threshold (3 points), belonging to the non-irritation level. The IS score of Comparative Example 1 group was 3.8, indicating mild irritation; Comparative Example 2 group was 2.4, and Comparative Example 3 group was 2.6, also indicating no irritation, but all higher than the present invention group. The positive control (commercially available 10% urea ointment) had an IS score of 4.5, indicating mild irritation. The IS score of the present invention group was significantly lower than that of Comparative Examples 1-3 and the positive control group (p<0.01), indicating that the composition of the present invention can effectively alleviate the irritation caused by urea.

[0088] Example 6 Efficacy verification of a guinea pig model of tinea pedis and chapped skin 1. Model Establishment Take a guinea pig, lightly rub the skin on its paws with sandpaper, and inoculate it with a suspension of Trichophyton rubrum (1×10⁻⁶). 8 (CFU / mL), administered once every 3 days for a total of 3 times. Starting 7 days after the last vaccination, apply 10% urea ointment daily to induce fissures for 7 consecutive days. Animals with obvious thickening of keratin and fissures 3-5 mm in length on their feet were selected for the experiment.

[0089] 2. Grouping and Dosing The successfully modeled guinea pigs were randomly divided into 3 groups of 10 each: Model control group: blank matrix (liquid crystal emulsion matrix without active ingredients) was applied. Positive control group: Apply commercially available 10% urea ointment; This invention relates to the application of the pharmaceutical composition prepared in Example 3.

[0090] Administer twice daily for 14 consecutive days.

[0091] 3. Evaluation Indicators (1) Crack healing rate: Measure the longest crack length and calculate the healing rate.

[0092] (2) Foot withdrawal threshold: The mechanical pain threshold was determined using Von Frey fibers.

[0093] (3) Erythema / irritation score: scored from 0 to 4 (0 points for no erythema, 4 points for very severe erythema).

[0094] (4) Fungal clearance rate: Microscopic examination and culture of skin scrapings from the affected area.

[0095] 4. Experimental Results Table 4. Efficacy results of the drug on day 14 in the guinea pig model of tinea pedis and tinea manuum with fissured skin. 5. Conclusion On day 14, the wound healing rate and foot retraction threshold of the present invention group were significantly better than those of the positive control group, and the erythema / irritation score was significantly lower than that of the positive control group, indicating that the composition of the present invention has excellent healing, analgesic and low irritation effects.

[0096] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing olive leaf extract, comprising the following steps: (1) Take dried olive leaves, crush them through a 20-40 mesh sieve, add 8-12 times the volume of 60%-80% ethanol, reflux extract at 50-70℃ 1-2 times, 1-2 hours each time, filter, and combine the filtrates; (2) Concentrate the filtrate under reduced pressure at a temperature below 50°C to obtain an extract with a relative density of 1.05-1.15; (3) The extract was purified by macroporous adsorption resin column. First, it was eluted with water, and then eluted with ethanol with a volume fraction of 30%-50%. The ethanol eluent was collected. (4) The ethanol eluent is concentrated and dried to obtain olive leaf extract, wherein the content of oleuropein is ≥20%.

2. A pharmaceutical composition for treating chapped skin caused by tinea pedis and manuum, characterized in that, It comprises the following components by weight percentage: urea 8%–12%; olive leaf extract 0.1%–3%; oat alkaloids 0.01%–0.5%; asiaticoside 0.05%–1%; shea butter 1%–8%; squalane 2%–10%; caprylic / capric triglycerides 3%–12%; cetearyl glucoside 0.5%–4%; and pharmaceutically acceptable carriers.

3. The pharmaceutical composition according to claim 2, characterized in that, The urea is selected from 8%, 9%, 10%, 11% or 12%; and / or the olive leaf extract is selected from 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5% or 3%; and / or the oat alkaloid is selected from 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4% or 0.5%; and / or the asiaticoside is selected from 0.05%, 0.1%, 0.2%, 0.3%, 0.5%, 0.8% or 1%.

4. The pharmaceutical composition according to claim 2, characterized in that, The shea butter is selected from 1%, 2%, 3%, 4%, 5%, 6%, 7% or 8%; and / or the squalane is selected from 2%, 4%, 6%, 8% or 10%; and / or the caprylic / capric triglyceride is selected from 3%, 5%, 7%, 9% or 12%; and / or the cetearyl glucoside is selected from 0.5%, 1%, 2%, 3% or 4%.

5. The pharmaceutical composition according to claim 2, characterized in that, The pharmaceutically acceptable carriers include one or more of the following: co-emulsifiers, humectants, preservatives, pH adjusters, and / or solvents; Preferably, the humectant is selected from one or more of glycerin, propylene glycol, butylene glycol, and sorbitol, and is used in an amount of 1% to 10%; and / or the preservative is selected from one or more of phenoxyethanol, p-hydroxyacetophenone, and ethylhexylglycerin, and is used in an amount of 0.1% to 1.0%; and / or the pH adjuster is selected from one or more of triethanolamine, citric acid, and sodium citrate; and / or the solvent is deionized water.

6. The pharmaceutical composition according to claim 5, characterized in that, The co-emulsifier is cetearyl alcohol, and / or the amount of the co-emulsifier is 1% to 4%.

7. The pharmaceutical composition according to claim 2, characterized in that, The preparation method of the olive leaf extract includes the following steps: (1) Take dried olive leaves, crush them through a 20-40 mesh sieve, add 8-12 times the volume of 60%-80% ethanol, reflux extract at 50-70℃ 1-2 times, 1-2 hours each time, filter, and combine the filtrates; (2) Concentrate the filtrate under reduced pressure at a temperature below 50°C to obtain an extract with a relative density of 1.05-1.15; (3) The extract was purified by macroporous adsorption resin column. First, it was eluted with water, and then eluted with ethanol with a volume fraction of 30%-50%. The ethanol eluent was collected. (4) The ethanol eluent is concentrated and dried to obtain olive leaf extract, wherein the content of oleuropein is ≥20%.

8. A method for preparing the pharmaceutical composition according to any one of claims 2-7, characterized in that, Includes the following steps: (1) Preparation of oil phase: Shea butter, squalane, caprylic / capric triglyceride, cetearyl glucoside, and optionally cetearyl alcohol are mixed and heated to 75-80℃ and stirred until completely melted to obtain the oil phase; (2) Preparation of aqueous phase: Deionized water, urea, humectant and preservative are mixed and heated to 75-80℃ and stirred until completely dissolved to obtain aqueous phase; (3) Emulsification: Under homogenization conditions, the aqueous phase is added to the oil phase and homogenized for 3-5 minutes to form a liquid crystal emulsion; (4) Cool down to below 45°C, add olive leaf extract, oat alkaloids and asiaticoside, and stir well; (5) Adjust the pH to 5.5-6.5 with a pH adjuster and cool to room temperature to obtain the final product.

9. Use of the pharmaceutical composition according to any one of claims 2-7 in the preparation of a medicament for treating chapped skin caused by tinea pedis.

10. The application according to claim 9, characterized in that, The drug is an ointment, cream, or gel.

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