Alkannin nasal spray film agent for repairing nasal mucosa injury and preparation method of shikonin nasal spray film agent

Abstract

The invention discloses an alkannin nasal spray film agent for repairing nasal mucosa lesion. The alkannin nasal spray film agent comprises 10 mg of L-alkannin; 1.5 g to 3 g of propylene glycol; 6 to 9 g of polyvinylpyrrolidone K30; 0.1 g to 0.4 g of methyl cellulose (MC); 80 [mu] l to 120 [mu] l of a solubilizer; 0.02 g to 0.08 g of a preservative; the solvent is a low-degree ethanol solution, and the mass fraction of the low-degree ethanol solution is 28-32%. The invention also discloses a preparation method of the film spraying agent. The shikonin film spraying agent can reduce the expression of proinflammatory factors, alleviate local inflammatory response and promote tissue remodeling by reducing the levels of inflammatory factors and chemotactic factors, and can generate a good treatment effect on nasal mucosa repair.

Description

Technical Field

[0001] The invention relates to a pharmaceutical composition for treating nasal mucosal damage, and in particular to a shikonin nasal spray for repairing nasal mucosal damage and a preparation method thereof. Background Art

[0002] Nasal mucosal wound repair is a process of local tissue inflammation, immune response, proliferation, and remodeling, which is completed by the participation of multiple cytokines and growth factors, but its molecular mechanism is still unclear. Among them, the inflammatory response is the key link in mucosal repair, and the growth factors and cytokines produced by it stimulate and coordinate the repair of new cells and tissues.

[0003] Clinically, antibiotics, hormones, nasal irrigants, and nasal sprays are commonly used to eliminate infection and reduce inflammation. A large number of studies have shown that conservative treatment of sinusitis has a poor cure rate and the condition is prone to recurrence. Endoscopic sinus surgery has a direct effect and a high cure rate. It can correct the patient's sinus anatomical abnormalities while reconstructing sinus ventilation and drainage, and has obvious clinical advantages. However, surgical operations will cause certain trauma to the sinuses and nasal cavity, leading to adverse conditions such as swelling of the surgical mucosa, nasal adhesions, and sinus ostium closure, which is not conducive to the recovery of postoperative nasal function and increases the risk of recurrence of chronic sinusitis. In addition, nasal mucosal damage can also be caused by trauma, radiotherapy, allergic reactions, and drug damage. Therefore, the use of adjuvant therapy to improve surgical prognosis and promote rapid recovery of the nasal mucosa has gradually become a hot topic in clinical research.

[0004] At present, most of the drugs directly used for the treatment of nasal diseases in clinical practice are smears and spray preparations. For example, erythromycin ointment, azelastine hydrochloride nasal spray, etc. Smear preparations can adhere well to the nasal mucosa, provide a moist microenvironment for the nasal cavity, and maintain a longer moist environment, but the drug application process is complicated. Relatively speaking, spray preparations are easy to apply, but they are directly applied to the nasal mucosa in the form of a spray, and there is a possibility that droplets will form in the nasal cavity and enter the throat and lower respiratory tract with the inhalation airflow, thereby causing discomfort to the patient. Spray film is a new drug delivery system that combines the advantages of film, spray and patch, which allows the drug to adhere to the lesion site while preventing the loss of the drug, and has the characteristics of convenient administration.

[0005] Spray film technology has been applied and proven in the medical field. Currently, most spray film agents mainly act on the skin surface, and there are also a small number of literatures that disclose that film-forming drugs can be applied to mucous membranes.

[0006] For example, Document 1: "Study on the Preparation Technology of Gentiana Macrophylla Sprayable Agent and Establishment of Its Quality Standards" published by Li Songyang et al.; Document 1 discloses:

[0007] (1) The types and dosages of film-forming excipients were screened based on film-forming time, volatilization amount, content uniformity, microscopic properties of the film, and viscosity; the proportion of glycerol was investigated based on moisture retention; the appropriate penetration enhancer and its dosage were screened based on the cumulative release of the active ingredient gentiopicroside; HPLC was used to determine the content of the active ingredient gentiopicroside in the Qinjiao spray, and a quality standard for the Qinjiao spray, including multiple inspection items such as moisture retention, was established. Results The film-forming formula was determined to be 6% polyvinylpyrrolidone K30 (PVP) and 0.2% methylcellulose MC-400 (MC), and the penetration enhancer formula was 2.5% propylene glycol and 2.5% azone; the quality standard of the gentiana spray was established as follows: the gentiana spray should contain no less than 0.76 mg / mL gentiopicroside; the moisture retention should be no less than 30.25%; the film-forming time should be no more than 240s; the volatile amount should be no less than 62.70%; the absorbance (A) value at 433nm after film formation should not be greater than 0.142, and the A value during film formation should not be less than 0.259; the kinematic viscosity should not be less than 6.91 mm2 / s; the sample pH value should be 3.67-5.50

[0008] (2) Take about 1.2g of the intermediate of Gentiana macrophylla in document 1, weigh it accurately, add 100mL of 45% ethanol, ultrasonically treat it for 20min (power 100W, frequency 40kHz), and filter it to obtain the intermediate solution of Gentiana macrophylla. , add different mass fractions of PVP, PVA, HPMC, and MC according to Table 2, investigate the types and dosage combinations of film-forming auxiliary materials, and comprehensively select the best film-forming material. Finally, four film-forming prescriptions of 7% PVP, 6% PVP+0.2% PVA, 6% PVP+0.2% MC, and 6% PVP+0.2% MC+0.2% PVA were preliminarily screened out. It can be seen from Table 5 of document 1 that the comprehensive scores of the four film-forming prescriptions are 89.79, 92.81, 94.03, and 90.6 respectively; it can be seen that the film-forming prescription using the combination of PVP+MC has better comprehensive performance.

[0009] The microscopic properties of the membrane are studied by using a microscope to study the membrane structure, which can visually observe the uniformity and density of the membrane after drug administration. The specific steps are to spray the drug solution evenly on a glass slide of the same size with the same number of sprays in an oven (37°C). After drying, observe the bubbles and number of the membrane under a 40x biological microscope. It can be seen from Table 3 of Reference 1 that although the film-forming formula using the combination of PVP+MC has better overall performance, the microscopic characteristics of the membrane are poor, the bubbles are large, and the bubble distribution is uneven; Reference 1 did not conduct relevant experiments on the optimization of the microstructure.

[0010] Document 2: Chinese Patent 201710242104.7 "An oral fast-dissolving film containing lutein albumin nanoparticles and its preparation method".

[0011] Document 2 discloses that lutein is prepared into lutein albumin nanoparticles by anti-solvent precipitation method, and then a solvent casting method is used to prepare an oral fast-dissolving film loaded with lutein albumin nanoparticles. The film has a complete surface without bubbles, uniform color, consistent thickness, and uniform drug distribution. The preparation method comprises the following steps:

[0012] The method comprises the following steps: a. dissolving lutein in tetrahydrofuran to a nearly saturated state as a good solvent and 0.4% albumin solution as a poor solvent, injecting the good solvent into the poor solvent under a 400W ultrasonic crushing condition, performing ice bath ultrasonication for 5 minutes, working for 3 seconds, and resting for 1 second to obtain a lutein albumin nano suspension; b. placing a polymer film-forming material in 85% ethanol to swell overnight to obtain a polymer solution; c. adding a plasticizer, a filler, a saliva stimulant and a flavoring agent to the polymer solution obtained in step b, and stirring evenly to obtain a film-forming solution; d. slowly dripping the film-forming solution obtained in step c into the lutein albumin nano suspension obtained in step a, stirring evenly to disperse it by magnetic force, standing it to defoam, pouring it on a gel template, drying it, and cutting it to obtain an oral quick-dissolving film carrying lutein albumin nanoparticles.

[0013] The polymer film-forming material in Document 2 is one of sodium carboxymethylcellulose, hydroxypropyl methylcellulose (HPMC), polyvinyl pyrrolidone, sodium alginate, fenugreek gum, and konjac gum, that is, without special restrictions on the film-forming material, the above method can still obtain a film-forming material with no bubbles on the surface and uniform thickness. Compared with Document 1, Document 2 adopts the method of defoaming and pouring the material on a gel wood board to obtain a quick-dissolving film, and then taking it orally. Document 1 adopts a spraying method to spray the drug directly onto the mucosa, which makes the film-forming material easier to be disordered when it reaches the mucosa. Therefore, there is a bubble problem in Document 1, but there is no bubble problem in Document 2; and the solvent selected in Document 2 is 85% ethanol solution.

[0014] Document 3: Chinese Patent 202410216993.X "A dexmedetomidine traditional Chinese medicine polysaccharide orodispersible film preparation, preparation method and application thereof".

[0015] Reference 3 uses polyvinyl pyrrolidone and traditional Chinese medicine polysaccharides as film-forming materials. The resulting orally dissolving film has a large specific surface area, excellent mechanical strength and flexibility, uniform drug distribution, and rapid evaporation of the solvent during the preparation process, effectively avoiding drug crystallization. In addition, this process is easier to achieve continuous production and has more stable quality.

[0016] In document 3, polyvinyl pyrrolidone and ethanol are mixed to obtain a polyvinyl pyrrolidone solution; the concentration of polyvinyl pyrrolidone in the polyvinyl pyrrolidone solution is 5% w / v to 10% w / v; polyvinyl pyrrolidone, traditional Chinese medicine polysaccharides, a pharmaceutically acceptable salt of dexmedetomidine and ethanol are mixed, and the ethanol used in the embodiment of document 3 is anhydrous ethanol.

[0017] From the prior art and the inventor's research, it can be known that a film-forming agent is a drug preparation suitable for acting on the skin and its mucous membranes. The film-forming quality of the film-forming agent includes film-forming time, volatilization amount, content uniformity, microscopic properties of the film, and viscosity; the formulation and preparation process of the pharmaceutical composition using the film-forming agent will affect the film-forming quality. It can be seen from document 1 that the film-forming prescription using the combination of PVP+MC has better comprehensive performance, but its microscopic characteristics are poor.

[0018] There are certain differences between mucous membrane and skin, which makes the requirements for film-forming quality not exactly the same. Mucous membrane is more sensitive to drugs than skin, so it is often not necessary to add penetration enhancers, but there are certain requirements for the comfort of medication, and irritation should be minimized. At the same time, it is not easy to directly observe the damage to the mucous membrane, so the pores and bubbles of the film-forming agent should be minimized as much as possible after film formation. The more bubbles there are, the less coverage and the more likely it is to produce leaks. These leaks cannot prevent the entry of external pathogenic microorganisms, which reduces the isolation effect of the film-forming agent and affects the treatment effect. Summary of the invention

[0019] The purpose of the present invention is to provide a shikonin nasal spray for repairing nasal mucosal damage and a preparation method thereof. The spray of the present invention can repair the nasal mucosa, and has few bubbles after film formation and high film quality.

[0020] To achieve the above object, one embodiment of the present invention provides a shikonin nasal spray for repairing nasal mucosal damage; each 100 ml of shikonin nasal spray includes the following materials:

[0021] Levorotatory shikonin 10mg; propylene glycol 1.5g~3g;

[0022] Polyvinyl pyrrolidone K30 6g~9g; Methyl cellulose MC 0.1g~0.4g;

[0023] Solubilizer 80ul~120ul; preservative 0.02g~0.08g;

[0024] The solvent is a low-degree ethanol solution, and the mass fraction of the low-degree ethanol solution is 28% to 32%.

[0025] Preferably, the solubilizing agent is any one of Tween-20, Tween-60, Tween-80, Poloxamer 407, Poloxamer 188, and hydrogenated castor oil polyoxyethylene ether.

[0026] Preferably, the preservative is one of ethylparaben, phenoxyethanol, diimidazole urea, methylisothiazolinone or sodium benzoate.

[0027] Preferably, each 100 ml of shikonin nasal spray film comprises the following materials:

[0028] L-shikonin 10mg; Propylene glycol 2g;

[0029] Polyvinylpyrrolidone K30 8g; Methylcellulose MC 0.1g;

[0030] Solubilizer 100ul; preservative 0.05g;

[0031] The solvent is a low-concentration ethanol solution, and the mass fraction of the low-concentration ethanol solution is 30%.

[0032] A method for preparing the shikonin nasal spray for repairing nasal mucosal damage as claimed in claim 1, the preparation method comprising the following steps:

[0033] Take a formula amount of L-shikonin and add it to an ethanol solution with a low solvent concentration, then add a solubilizer and ultrasonically dissolve it; after ultrasonically dissolving, add a formula amount of polyvinyl pyrrolidone K30 and methyl cellulose MC and stir to dissolve; then take a formula amount of propylene glycol and a preservative and add them thereto, stir and then add a solvent to make up the volume.

[0034] In summary, the present invention has the following advantages:

[0035] 1. Shikonin spray can reduce the expression of pro-inflammatory factors, alleviate local inflammatory response and promote tissue remodeling by reducing the levels of inflammatory factors and chemokines, and can produce a good therapeutic effect on nasal mucosa repair.

[0036] 2. The present invention adopts a film-forming formula of PVP+MC to obtain good comprehensive performance, and at the same time reduces the concentration of ethanol, so that the irritation of the film-forming agent to the human nasal cavity is reduced, and the administration of the film-forming agent to patients with nasal diseases in the form of a spray film can bring more comfort to the patients. At the same time, a good microstructure can be obtained, and the generation of bubbles can be reduced.

[0037] The benefits of less bubbles:

[0038] (1) The film formed by the spray film is more uniform and smooth after use. If there are too many bubbles, the film surface will be uneven. The spray film with fewer bubbles can be more evenly covered on the target surface when used, ensuring that all parts can be effectively protected or treated.

[0039] (2) Good barrier properties: Spray agents with fewer bubbles can form a denser film structure, have better barrier properties against oxygen, moisture, etc., and are more conducive to the repair of the nasal mucosa.

[0040] (3) Strong adhesion: Fewer bubbles can make the contact between the spray film and the substrate closer, thereby improving the adhesion of the film; the presence of bubbles may cause uneven mechanical properties of the film.

[0041] 3. The film-forming agent combination of PVP-MC used in the present invention is highly safe and non-irritating, and is suitable for nasal use; in addition, PCP has good solubility in both water and ethanol, and can stably dissolve in 30% ethanol. Compared with the use of chitosan as a film-forming material, chitosan itself has antibacterial properties, high price, and low solubility in 30% ethanol. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 A histopathological picture under a microscope in one embodiment of the present invention;

[0043] Figure 2 The expression of Occludin and ZO-1 proteins in different groups was detected by Western Blot in one embodiment of the present invention;

[0044] Figure 3 The blood routine test results of each group of rabbits on the 7th day in one embodiment of the present invention;

[0045] Figure 4 The levels of inflammatory factors in the nasal mucosal wound tissue of each group of rabbits in one embodiment of the present invention;

[0046] Figure 5 The film microscopic properties of Example 1 and Experimental Examples 31 to 33 of the present invention are shown;

[0047] Figure 6 The film microscopic properties of Example 1 and Experimental Examples 41 and 42 of the present invention are shown;

[0048] Figure 7 The film microscopic properties of Experimental Examples 431 and 432 of the present invention are shown;

[0049] Figure 8 These are the film microscopic properties of Experimental Examples 441 and 442 of the present invention. DETAILED DESCRIPTION

[0050] The present invention provides a shikonin nasal spray for repairing nasal mucosal damage. Each 100 ml of the shikonin nasal spray comprises the following materials:

[0051] Levorotatory shikonin 10mg; propylene glycol 1.5g~3g;

[0052] Polyvinyl pyrrolidone K30 6g~9g; Methyl cellulose MC 0.1g~0.4g;

[0053] Solubilizer 80ul~120ul; preservative 0.02g~0.08g;

[0054] The solvent is a low-degree ethanol solution, and the mass fraction of the low-degree ethanol solution is 28% to 32%.

[0055] The solubilizer is any one of Tween-20, Tween-60, Tween-80, poloxamer 407, poloxamer 188, and hydrogenated castor oil polyoxyethylene ether. The preservative is one of ethylparaben, phenoxyethanol, diimidazole urea, methylisothiazolinone, or sodium benzoate.

[0056] Example 1

[0057] A shikonin nasal spray for repairing nasal mucosal damage, each 100 ml of shikonin nasal spray comprises the following materials:

[0058] L-shikonin 10mg; Propylene glycol 2g;

[0059] Polyvinylpyrrolidone K30 8g; Methylcellulose MC 0.1g;

[0060] Solubilizer 100ul; preservative 0.05g;

[0061] The solvent is a low-concentration ethanol solution, and the mass fraction of the low-concentration ethanol solution is 30%.

[0062] The preparation method comprises the following steps:

[0063] Take a formula amount of L-shikonin and add it to an ethanol solution with a low solvent concentration, then add a solubilizer and ultrasonically dissolve it; after ultrasonically dissolving, add a formula amount of polyvinyl pyrrolidone K30 and methyl cellulose MC and stir to dissolve; then take a formula amount of propylene glycol and a preservative and add them thereto, stir and then add a solvent to make up the volume.

[0064] Experiment 1: Pharmacological experiment of shikonin nasal spray

[0065] 1. Reagents

[0066] Shikonin (HPLC grade, purity ≥98%) was purchased from the National Institute for Drug and Biological Products Control;

[0067] Erythromycin ointment (1% × 10 g) is a product of Shanghai General Pharmaceutical Co., Ltd.;

[0068] Polyvinyl pyrrolidone K30 and methyl cellulose MC-400 were purchased from Chengdu Kelong Chemical Co., Ltd.;

[0069] Ethyl paraben was purchased from Hunan Erkang Pharmaceutical Co., Ltd.;

[0070] Methanol (AR, HPLC), acetonitrile (AR, HPLC), and all other chemicals were purchased from Thermo Fisher Scientific.

[0071] Tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin 6 (IL-6) Elisa kits were purchased from Jiangsu ELISA Industrial Co., Ltd.;

[0072] The reagents used in western blotting experiments, including 5% BSA blocking solution, were purchased from Shanghai Xiabukang Biotechnology Co., Ltd.;

[0073] RNA extraction kit was purchased from Qiagen, Germany;

[0074] The reverse transcription kit was purchased from Tiangen Biochemical Technology Co., Ltd.; the fluorescence quantitative reagent was purchased from Baoriyi Biotechnology Co., Ltd.

[0075] 2. Experimental Animals

[0076] Three-month-old female New Zealand white rabbits weighing 2.0-3.0 kg were selected for the experiment. Each rabbit was housed in a cage at 25±2℃, 60-65% humidity, 12h light-dark alternation, and fed with conventional feed and free access to water.

[0077] 3. Experimental methods

[0078] (1) Establish a nasal mucosal mechanical injury model; rabbits were randomly divided into 4 groups, namely, control group, model group, shikonin nasal spray group (100 μg / mL), and erythromycin ointment group. The model rabbits were given medication at a fixed time once a day for 7 consecutive days. After the experiment, all rabbits were euthanized and nasal mucosal samples were quickly collected for subsequent analysis.

[0079] (2) Inspection items

[0080] 2.1. Histopathological analysis: Histopathological changes were observed and photographed under an Eclipse CI microscope (Nikon, Japan).

[0081] 2.2. Western Blot: The rabbit nasal mucosal tissue protein of each group was extracted and the protein concentration was determined, followed by SDS-PAGE, protein blot observation, and the optical density value of the target band was analyzed using ImageJ software.

[0082] 2.3. Hematological analysis: The white blood cell count (WBC), lymphocyte count (LYM), red blood cell count (RBC), platelet count (PLT), neutrophil percentage (Gran%) and hemoglobin concentration (HGB) were analyzed using a fully automatic blood cell analyzer.

[0083] 2.4. Enzyme-linked immunosorbent assay: Nasal mucosal tissues of rabbits in each group were obtained and the contents of TNF-α, IL-1β and IL-6 therein were determined according to the instructions of the Elisa kit.

[0084] 4. Experimental results

[0085] 4.1 Effect of shikonin nasal spray on wound healing in rabbits with nasal mucosal injury

[0086] See also Figure 1 , Figure 1 Part A shows the healing of nasal mucosal injuries in rabbits of different groups. Figure 1 Part B shows the results of histopathological observation of rabbit nasal mucosa. The rabbit model of nasal mucosal injury was used to evaluate the effect of shikonin spray. Histopathological examination showed that after 7 days of shikonin nasal spray, the epithelium of the rabbit nasal mucosa was neatly arranged, the loss of cilia and the infiltration of inflammatory cells were reduced, and there was no obvious expansion of the blood vessels in the lamina propria.

[0087] 4.2 Effect of shikonin nasal spray on the expression levels of tight junction proteins occludin and ZO-1 in nasal mucosa of model rabbits

[0088] refer to Figure 2 Occludin and ZO-1 work together in the barrier function of the cell layer to ensure the integrity and selective permeability of the cell layer, which is essential for maintaining the normal function and morphology of the tissue. The expression levels of tight junction proteins Occludin and ZO-1 in rabbit nasal mucosal tissue were detected by western blot. The study showed that Occludin and ZO-1 in the SNSF group were significantly higher than those in the model group (p < 0.05, p < 0.01), indicating that SNSF helps to enhance the barrier function of the rabbit nasal mucosa and improve the tissue remodeling of the lost mucosa.

[0089] 4.3 Effects of Shikonin Nasal Spray on Blood Physiological Parameters in Model Rabbits

[0090] like Figure 3As shown in the figure, the results of blood routine tests of rabbits showed that on the 7th day of modeling, compared with the control group, the WBC, Gran%, PLT, and Lym of the rabbits in the model group were significantly increased (p<0.05, p<0.01), and HGB and RBC were significantly decreased (p<0.05, p<0.01). After SNSF treatment, WBC, Gran%, PLT, and Lym were significantly decreased (p<0.05, p<0.01), and HGB and RBC were significantly increased (p<0.05, p<0.01). This indicates that mechanical trauma to the nasal mucosa of the rabbits in the model group caused a significant inflammatory response and reduced immunity. The decrease in HGB and RBC indicated that the trauma caused anemia in the body. Shikonin nasal spray can improve inflammation and reduce anemia after treatment, and the therapeutic effect is not significantly different from erythromycin.

[0091] 4.4 Effect of Shikonin Nasal Spray on Inflammation Levels in Model Rabbits

[0092] refer to Figure 4 Compared with the control group, the levels of IL-1β, IL-6 and TNF-α in the model group increased significantly (p<0.01), and compared with the model group, the levels of IL-1β, IL-6 and TNF-α in the SNSF group decreased significantly (p<0.01). The results showed that SNSF significantly reduced the levels of pro-inflammatory factors in nasal mucosal tissue.

[0093] In summary, it can be seen from the above experimental results that the shikonin spray film of the present invention can produce a good therapeutic effect on nasal mucosa repair.

[0094] Example 2

[0095] A shikonin nasal spray for repairing nasal mucosal damage, each 100 ml of shikonin nasal spray comprises the following materials:

[0096] L-shikonin 10mg; Propylene glycol 2.5g;

[0097] Polyvinylpyrrolidone K30 7g; Methylcellulose MC 0.32g;

[0098] Solubilizer Tween-80 90ul; preservative ethylparaben 0.03g;

[0099] The solvent is a low-concentration ethanol solution, and the mass fraction of the low-concentration ethanol solution is 30%.

[0100] The preparation method comprises the following steps:

[0101] Take a formula amount of L-shikonin and add it to an ethanol solution with a low solvent concentration, then add a solubilizer and ultrasonically dissolve it; after ultrasonically dissolving, add a formula amount of polyvinyl pyrrolidone K30 and methyl cellulose MC and stir to dissolve; then take a formula amount of propylene glycol and a preservative and add them thereto, stir and then add a solvent to make up the volume.

[0102] Example 3

[0103] A shikonin nasal spray for repairing nasal mucosal damage, each 100 ml of shikonin nasal spray comprises the following materials:

[0104] L-shikonin 10mg; Propylene glycol 2.0g;

[0105] Polyvinylpyrrolidone K30 6g; Methylcellulose MC 0.2g;

[0106] Solubilizer Tween-80 80ul; preservative ethylparaben 0.02g;

[0107] The solvent is a low-concentration ethanol solution, and the mass fraction of the low-concentration ethanol solution is 30%.

[0108] The preparation method comprises the following steps:

[0109] Take a formula amount of L-shikonin and add it to an ethanol solution with a low solvent concentration, then add a solubilizer and ultrasonically dissolve it; after ultrasonically dissolving, add a formula amount of polyvinyl pyrrolidone K30 and methyl cellulose MC and stir to dissolve; then take a formula amount of propylene glycol and a preservative and add them thereto, stir and then add a solvent to make up the volume.

[0110] Example 4

[0111] A shikonin nasal spray for repairing nasal mucosal damage, each 100 ml of shikonin nasal spray comprises the following materials:

[0112] L-shikonin 10mg; Propylene glycol 3g;

[0113] Polyvinylpyrrolidone K30 9g; Methylcellulose MC 0.4g;

[0114] Solubilizer Tween-80 120ul; preservative ethylparaben 0.08g;

[0115] The solvent is a low-concentration ethanol solution, and the mass fraction of the low-concentration ethanol solution is 32%.

[0116] The preparation method comprises the following steps:

[0117] Take a formula amount of L-shikonin and add it to an ethanol solution with a low solvent concentration, then add a solubilizer and ultrasonically dissolve it; after ultrasonically dissolving, add a formula amount of polyvinyl pyrrolidone K30 and methyl cellulose MC and stir to dissolve; then take a formula amount of propylene glycol and a preservative and add them thereto, stir and then add a solvent to make up the volume.

[0118] Example 5

[0119] A shikonin nasal spray for repairing nasal mucosal damage, each 100 ml of shikonin nasal spray comprises the following materials:

[0120] L-shikonin 10mg; Propylene glycol 2.4g;

[0121] Polyvinylpyrrolidone K30 7.5g; Methylcellulose MC 0.30g;

[0122] Solubilizer Tween-80 100ul; preservative ethylparaben 0.06g;

[0123] The solvent is a low-concentration ethanol solution, and the mass fraction of the low-concentration ethanol solution is 29%.

[0124] The preparation method comprises the following steps:

[0125] Take a formula amount of L-shikonin and add it to an ethanol solution with a low solvent concentration, then add a solubilizer and ultrasonically dissolve it; after ultrasonically dissolving, add a formula amount of polyvinyl pyrrolidone K30 and methyl cellulose MC and stir to dissolve; then take a formula amount of propylene glycol and a preservative and add them thereto, stir and then add a solvent to make up the volume.

[0126] Experiment 2: Film-forming process experiment of shikonin film-spraying agent of the present invention

[0127] 1. Experimental instruments

[0128]

[0129] 2. Experimental Materials

[0130] name source L-Shikonin China Inspection and Quarantine Agency Polyvinylpyrrolidone K30 Chengdu Cologne Methylcellulose MC-400 Chengdu Cologne Ethylparaben Hunan Erkang Pharmaceutical Methanol Thermo Fisher Scientific Acetonitrile Thermo Fisher Scientific

[0131] 3. Ethanol concentration experiment

[0132] L-shikonin is a fat-soluble component, easily soluble in high-concentration ethanol or petroleum ether ethyl acetate and other fat-soluble solvents, but insoluble in water. Considering that shikonin spray is mainly used for nasal application, the concentration of ethanol needs to be controlled to avoid irritation.

[0133] Take 10 mg of laboratory levorotatory shikonin, add anhydrous ethanol to make up to 10 ml, and prepare a mother solution of 1 mg / ml.

[0134] Take 1 ml of the mother solution and add water to make up to 10 ml to prepare a 0.1 mg / ml solution with an ethanol concentration of 10%;

[0135] Take 1 ml of the mother solution, add 1 ml of anhydrous ethanol and then add water to make up to 10 ml to prepare a 0.1 mg / ml solution with an ethanol concentration of 20%;

[0136] Take 1 ml of the mother solution, add 2 ml of anhydrous ethanol and then add water to make up to 10 ml to prepare a 0.1 mg / ml solution with an ethanol concentration of 30%;

[0137] Take 1 ml of the mother solution, add 3 ml of anhydrous ethanol and then add water to make up to 10 ml to prepare a 0.1 mg / ml solution with an ethanol concentration of 40%;

[0138] Observe the color change. The color of 10%-30% ethanol concentration gradually deepens, and the colors of 30% and 40% are basically the same. After standing overnight, 30% has very little precipitation, and 40% has no precipitation. 10% and 20% have floccules. Take the supernatant to determine the content of L-shikonin. The test results are shown in the following table:

[0139] Table 1: Ethanol concentration experimental results

[0140] Ethanol concentration Solution color Ethanol odor precipitation content Recovery rate / % 10％ Pink Mild taste More precipitation 0.0322 3.22 20％ Pink Mild taste There is precipitation 0.1222 12.22 30％ Claret Mild taste Very little precipitation 0.9656 96.56 40％ Claret Mild taste No precipitation 0.9785 97.85

[0141] It can be seen from the above experimental results that when the ethanol concentration is too low, precipitation will occur and the recovery rate will be greatly reduced. When the ethanol concentration is above 30%, the recovery rate or content can meet the medication requirements and there is very little precipitation.

[0142] 4. Film-forming material experiment

[0143] In most cases, without considering special circumstances, the solution viscosity of the selected film-forming material should be small and the film should be formed quickly, so that the spraying agent can form a spray state and form a good film. The experiment of film-forming materials is divided into two parts, one is the performance of a single film-forming material, and the other is the performance of a composite film-forming material.

[0144] The experimental results of the film-forming performance of the single film-forming material of the present invention are shown in Table 2:

[0145] Table 2: Film-forming properties of single film-forming materials

[0146]

[0147] The film-forming performance test results of the composite film-forming material of the present invention are shown in Table 3:

[0148] Table 3: Film-forming properties of composite film-forming materials

[0149] Film-forming materials Solution state Spray state Film formation 8% PVP-30 + MC 0.1% Low viscosity, completely dissolved, no precipitation Fog Film formation (16min) 8% PVP-30 + MC 0.2% Low viscosity, completely dissolved, no precipitation Fog Film formation (16min) 8%PVP-30+MC 0.3% Low viscosity, completely dissolved, no precipitation Half spray Film formation (16min) 8%PVP-30+MC 0.4% Low viscosity, completely dissolved, no precipitation Half spray Film formation (16min) 8%PVP-30+MC 0.5% Low viscosity, small amount of floccules Half spray Film formation (16min)

[0150] 5. Propylene glycol dosage experiment

[0151] Table 4: Test results of different propylene glycol amounts

[0152] Propylene glycol (ml) Solution state Spray state Film formation 1 Low viscosity, completely dissolved, no precipitation Fog Film formation (13min) 2 Low viscosity, completely dissolved, no precipitation Fog Film formation (17min) 3 Low viscosity, completely dissolved, no precipitation Fog Film formation (21min) 4 Low viscosity, completely dissolved, no precipitation Fog Film formation (28min) 5 Low viscosity, completely dissolved, no precipitation Half spray Film formation (33min)

[0153] 6. Orthogonal experiment

[0154] 6.1. Select three factors that have the greatest impact on the film-forming effect: PVP-30 amount (A), MC amount (B), and propylene glycol amount (C). Select three levels for each factor to design an L9 orthogonal experiment. The specific factor level arrangement is shown in Table 5.

[0155] Table 5: Orthogonal test factor level arrangement table

[0156] Factor Levels PVP-30 dosage MC dosage Propylene glycol dosage 1 5 0.1 1 2 8 0.2 2 3 10 0.3 3

[0157] The present invention takes film-forming time, spray effect, film toughness, adhesion and solution state as evaluation indicators. According to the influence of each evaluation factor on the film-forming effect, the weight coefficient of film-forming time is set to 0.3, the spray effect is 0.1, the film toughness is 0.2, the film microscopic properties are 0.2, the adhesion is 0.1, and the solution state is 0.1. The scoring standards are set to optimize the best formula process.

[0158] 6.2 Evaluation Method

[0159] Film-forming time: spray the film-forming agent evenly on the glass slide until the film is no longer sticky) Total score: 30 points, minus 2 points for every additional minute.

[0160] Spray effect: The full score is 10 points, the best is to spray a uniform mist of liquid, with a score of 8-10 points; droplet-like is second, with a score of 5-7 points; line-like is the worst, with a score below 5 points.

[0161] Microscopic properties of membrane: spray the solution evenly on a glass slide of the same size with the same number of sprays, and place in an oven (37°C). After drying, observe the bubbles and quantity of the membrane under a biological microscope at 40 times magnification, with a full score of 20 points, very few bubbles 18-20, a few bubbles 15-17, many bubbles 11-14, and very many bubbles 0-9.

[0162] Film toughness:

[0163] The full score is 20 points. The film is cut into a square with a certain side length (5cm), rolled into a uniform strip along the side length, the starting length is recorded, and the self-made stretching device is used to stretch it. The length of the film at the moment of rupture is recorded. The length at the moment of rupture minus the starting length is the stretched length of the film. If the stretched length is 4 times or more of the starting length, it will be 20 points. If the stretched length is 4 times the starting length and does not decrease by one time, 5 points will be deducted.

[0164] Adhesion: The film should not stick to clothes after forming. Press cotton lightly on the surface of the film, then remove the cotton and observe the adhesion of the surface to the cotton fibers. The full score is 10 points, very little cotton fiber 10 points, less fiber 5-9 points, more fiber 0-5 points.

[0165] Solution state: Place 100 ml of the spray agent in a beaker and observe whether there is precipitation or floccules. The full score is 10 points, very little precipitation or floccules 8-10 points, a small amount of precipitation or floccules 5-7 points, and a large amount of precipitation or floccules 0-4 points.

[0166] 6.3. Orthogonal Experiment Evaluation Results

[0167]

[0168]

[0169] By analyzing the orthogonal experimental evaluation results, the optimal process recipe of the present invention can be finally obtained:

[0170] L-shikonin 10mg; Propylene glycol 2g;

[0171] Polyvinylpyrrolidone K30 8g; Methylcellulose MC 0.1g;

[0172] Solubilizer 100ul; preservative 0.05g;

[0173] The solvent is a low-concentration ethanol solution, and the mass fraction of the low-concentration ethanol solution is 30%.

[0174] 7. Sample testing

[0175] The shikonin nasal spray in Example 1 of the present invention was subjected to quality inspection in three batches, and the specific inspection results are shown below.

[0176] Detection Type Test results pH 4.0 Film forming time 22min Moisture absorption rate 27.5％

[0177] 8. Spraying agent stability test

[0178] Accurately pipette 2 ml of the sample of lithospermum spray prepared by the optimal process, make it into the test solution, and place it at room temperature. Take the supernatant on 0d, 1d, 2d, 3d, 4d, and 5d, respectively, and treat the samples according to the chromatographic method. Before sampling on the 6th day, ultrasonic mixing is performed, and then samples are taken and treated according to the chromatographic method to determine the peak area.

[0179]

[0180]

[0181] The results show that the content of the lithospermum spray agent gradually decreases and then tends to be stable as the storage time increases. Since shikonin gradually precipitates during the storage process, it needs to be shaken well before use.

[0182] Experiment 3: Study on the effects of components of shikonin nasal spray on film-forming bubbles

[0183] In Experiment 3, each experimental group was prepared according to 100 ml of spray film, and the preparation method was as described in the example. Based on Example 1, the same film-forming agent components were used, and the ethanol concentration was adjusted separately to test the effect of different ethanol concentrations on film-forming bubbles. The components of the shikonin film-forming agent of each experimental group are as follows:

[0184]

[0185]

[0186] The film sprays prepared in Example 1 to Experimental Example 33 were tested for film microscopic properties. The test method was as follows: the liquid was sprayed evenly on a glass slide of the same size with the same number of sprays, placed in a 37°C oven for treatment, and after drying, the film was placed under a biological microscope at 40 times to observe the bubbles and quantity. The results of the observations were Figure 5 As shown; Figure 5 A1 is the film microscopic properties of Example 1, A31 is the film microscopic properties of Experimental Example 31, A32 is the film microscopic properties of Experimental Example 32, and A33 is the film microscopic properties of Experimental Example 33. Compared with the other three experimental examples, Example 1 uses a lower solvent concentration, that is, a low-degree ethanol solution with a mass fraction lower than 32%. The ethanol concentration in Experimental Examples 31 to 33 gradually increases, and the mass fraction is greater than 42%. It can be seen from the test results that the bubbles in Example 1 are the least and the number is good; under the same circumstances, when the ethanol concentration increases, the number and size of bubbles in the film gradually increase, and the number and size of bubbles reach the maximum when anhydrous ethanol is used. It can be seen that in order to reduce the irritation to the nasal mucosa, the present invention chooses to reduce the ethanol concentration and adopts an ethanol solution with a mass fraction lower than 32%, and unexpectedly obtains the effect of reducing the number and size of bubbles.

[0187] Experiment 4: Study on the influence of different film-forming agent combinations on film-forming bubbles

[0188] Several different film-forming agent components from those in Example 1 were used, and each film-forming agent was tested using different ethanol concentrations to study the effects of different ethanol concentrations and different film-forming agents on film-forming bubbles. The composition of each experimental example in Experiment 4 is as follows:

[0189]

[0190]

[0191] The film spraying agents prepared in all embodiments or experimental examples in Experiment 4 were tested for film microscopic properties. The testing method was as follows: the liquid was evenly sprayed on a glass slide of the same size with the same number of sprays, placed in a 37°C oven for treatment, and after drying, the film was placed under a 40x biological microscope to observe the bubbles and quantity. The test results are shown in the attached figure.

[0192] Figure 6 1 is an observation diagram of the film microscopic properties of Example 1, Experimental Example 41, and Experimental Example 42. A1 is the film microscopic properties of Example 1, A41 is the film microscopic properties of Experimental Example 41, and A42 is the film microscopic properties of Experimental Example 42. Figure 6 It can be seen that the film formation conditions of Example 1, Experimental Example 41 and Experimental Example 42 are relatively similar, and there is no significant difference in the bubble size and bubble quantity among the three. This shows that the change of some auxiliary materials has very little effect on the bubbles.

[0193] Experimental Examples 431 and 432 used different film-forming agents from Example 1; the film-forming agent components of the two experimental examples were the same, but the mass fractions of the solvent ethanol used were different. Figure 7 As shown, the microscopic properties observation pictures of the films of Experimental Example 431 and Experimental Example 432 are A431 and A432 respectively; the difference in the size and number of bubbles in the films is very small, indicating that when PVA is used as the film-forming agent, the significant change in ethanol concentration has very little effect on the film-forming bubbles and there is no significant difference.

[0194] Similarly, Experimental Examples 441 and 442 used different film-forming agents from Example 1; the film-forming agent components of the two experimental examples were the same, but the mass fractions of the solvent ethanol used were different. Figure 8 As shown, the microscopic properties observation images of the films of Experimental Example 441 and Experimental Example 442 are A441 and A442, respectively.

[0195] like Figure 8 As shown; the bubbles in Experimental Examples 441 and 442 are larger than those in Experimental Examples 431 and 432; however, the difference in size and number of the membrane bubbles between Experimental Examples 441 and 442 is very small, indicating that when a combination of PVA and HPMC is used as a film-forming agent, the significant change in ethanol concentration has very little effect on the film-forming bubbles and there is no significant difference.

[0196] Although the specific implementation of the present invention is described in detail in conjunction with the drawings, it should not be understood as limiting the scope of protection of this patent. Within the scope described in the claims, various modifications and variations that can be made by those skilled in the art without creative work still fall within the scope of protection of this patent.

Claims

1. A shikonin nasal spray for repairing nasal mucosal damage, characterized in that: Each 100ml of Shikonin nasal spray contains the following materials: Levorotatory shikonin 10mg; propylene glycol 1.5g~3g; Polyvinyl pyrrolidone K30 6g~9g; Methyl cellulose MC 0.1g~0.4g; Solubilizer 80ul~120ul; preservative 0.02g~0.08g; The solvent is a low-degree ethanol solution, and the mass fraction of the low-degree ethanol solution is 28% to 32%.

2. The shikonin nasal spray for repairing nasal mucosal damage according to claim 1, characterized in that: The solubilizing agent is any one of Tween-20, Tween-60, Tween-80, Poloxamer 407, Poloxamer 188, and hydrogenated castor oil polyoxyethylene ether.

3. The shikonin nasal spray for repairing nasal mucosal damage according to claim 1, characterized in that: The preservative is one of ethylparaben, phenoxyethanol, diimidazole urea, methylisothiazolinone or sodium benzoate.

4. The shikonin nasal spray for repairing nasal mucosal damage according to claim 1, characterized in that: Each 100ml of Shikonin nasal spray contains the following materials: L-shikonin 10mg; Propylene glycol 2g; Polyvinylpyrrolidone K30 8g; Methylcellulose MC 0.1g; Solubilizer 100ul; preservative 0.05g; The solvent is a low-concentration ethanol solution, and the mass fraction of the low-concentration ethanol solution is 30%.

5. A method for preparing the shikonin nasal spray for repairing nasal mucosal damage as described in any one of claims 1 to 4, characterized in that: The preparation method comprises the following steps: Take a formula amount of L-shikonin and add it to an ethanol solution with a low solvent concentration, then add a solubilizer and ultrasonically dissolve it; after ultrasonically dissolving, add a formula amount of polyvinyl pyrrolidone K30 and methyl cellulose MC and stir to dissolve; then take a formula amount of propylene glycol and a preservative and add them thereto, stir and then add a solvent to make up the volume.

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