A hemostatic spray film and its preparation method

Abstract

The present invention relates to a film spray, and more particularly to a hemostatic film spray and a preparation method thereof. The hemostatic film spray is characterized by comprising a Panax notoginseng extract, a film-forming material, a film-forming solvent, glycerol, and water. In the preparation method of the present invention, mixing is performed at room temperature, without the need for high temperatures, thereby ensuring that the notoginseng in the Panax notoginseng extract is not destroyed. The film is also stable in the film-forming solvent, with each component fully dissolved without precipitation. The fine mist formed by the film of the present invention is thin, easy to tear, has good toughness, a long film duration, high transparency, high uniformity, and a moderate thickness, with a comprehensive score of 90 or above.

Description

Technical Field

[0001] The present invention relates to a film spray, in particular to a hemostatic film spray and a preparation method thereof Background Art

[0002] Spray film is a topical drug preparation that combines the dosage form characteristics of film, spray and patch. It is administered simply and quickly using a spray bottle. After being applied to the wound area, the solvent evaporates rapidly to form a drug film. While continuously releasing the loaded drug, it isolates the wound surface, promotes wound healing and prevents wound infection.

[0003] Film-forming materials are the key components of spray film formation. They need to be stable, non-toxic, non-irritating, non-teratogenic and non-carcinogenic; have good film-forming and demolding properties, and be able to form a film with sufficient strength and flexibility; they also need to have stable chemical properties and not interfere with the therapeutic effects of the main ingredients and other auxiliary materials; considering the prospects for industrialization, they also need to be abundant in source and cheap.

[0004] Common film-forming materials can be divided into two categories: natural polymer film-forming materials and synthetic polymer film-forming materials. Natural polymer film-forming materials include: chitosan and chitosan hydrochloride [1], sodium alginate, Bletilla striata, etc. [2]. Synthetic polymer film-forming materials include: polyvinyl alcohol (including polyvinyl alcohol PVA compounds and polyvinyl acetal (PVB) compounds obtained by condensation with aldehyde compounds, and polyvinyl pyrrolidone PVP), acrylic copolymers (including acrylates and carbomers), cellulose derivatives (including ethyl cellulose (EC), cellulose acetate (CA), hydroxypropyl cellulose (HPC), nitrocellulose (NC), etc.), polyurethane and polyamide (PA) [2][3].

[0005] Panax notoginseng is the dried root and rhizome of the perennial herbaceous plant Panax notoginseng (Burk.) FHChen, belonging to the Araliaceae family. The Chinese Pharmacopoeia states that it has the functions of “dispersing blood stasis, stopping bleeding, reducing swelling and relieving pain” [4]. In clinical practice, small doses of Panax notoginseng powder are often used as a hemostatic drug. Small doses of Panax notoginseng stop bleeding, while large doses promote blood circulation.

[0006] The main hemostatic components of Panax notoginseng are dencichine and quercetin, of which dencichine is the main one. Dencichine is a special amino acid that can promote platelet aggregation, deformation, release of ADP, platelet factor III and Ca 2 + and other substances, shortening the bleeding and coagulation time, thus achieving a hemostatic effect. However, due to the instability of notoginseng, which is easily destroyed after heating, the hemostatic effect of notoginseng is generally achieved by using it raw [5].

[0007] However, as a traditional Chinese medicine extract (Sanqi), it will encounter the following problems:

[0008] 1. Film-forming materials themselves are easy to form, but the resulting films generally have poor toughness. This is a common problem that needs to be addressed in film formulation design. The performance of the film can be optimized by adding ingredients such as glycerol. However, hemostatic spray films with Panax notoginseng extract as the active ingredient have poor film-forming properties and low toughness during the preparation process due to the presence of peptides, polysaccharides, and saponins in the extract. Furthermore, the films are unstable and may precipitate during use. Therefore, when screening excipients and optimizing formulations for Chinese herbal extracts, it is not possible to simply use commonly used components and ratios; a more complex screening process is required.

[0009] 2. When the volatile organic solvent is insufficient, the film-forming time will be increased, but excessive organic solvent will cause the precipitation of the film-forming material. It is necessary to find the optimal ratio to ensure good solubility of the components while forming the film faster;

[0010] 3. The film-forming material dissolves slowly and generally requires a high temperature of more than 80°C to dissolve. The hemostatic components of Panax notoginseng are easily destroyed at high temperatures, resulting in the inability to prepare the film by conventional operations. Summary of the Invention

[0011] Technical issues to be solved:

[0012] Aiming at the technical problems such as poor film formation, low toughness and unstable film when preparing film preparations with Panax notoginseng extract, a new film preparation is provided, which effectively overcomes the above problems.

[0013] Technical solution:

[0014] A hemostatic spray film, characterized by containing notoginseng extract, film-forming material, film-forming solvent, glycerol and water.

[0015] The Panax notoginseng extract is prepared by the following steps: weighing 100 g of coarse Panax notoginseng medicinal material particles, soaking them in an equal volume of corresponding solvent for 24 hours, extracting them by ultrasonication of alcohol solution, heating of alcohol solution, ultrasonication of aqueous solution or heating of aqueous solution, filtering, combining the filtrate, and freeze-drying the aqueous extract.

[0016] The ultrasonic conditions are as follows: ultrasonic frequency of 40 kHz, power of 180 W, ultrasonication for 20 min / time, filtration, and addition of more than 2 times the volume of solvent for ultrasonic extraction;

[0017] Heating conditions: 60℃ water bath, 20min / time, filtration, adding more than 2 times the volume of solvent, and heating extraction.

[0018] The film-forming material is polyvinyl alcohol or a mixture of polyvinyl alcohol and polyvinyl pyrrolidone. The film-forming solvent is ethanol, isopropyl alcohol, ethyl acetate or a mixed solvent of any two of the above.

[0019] The film-forming material is a mixture of PVP-K30 and PVA-0588.

[0020] A hemostatic spray film, characterized in that, by mass percentage, the formula is 1.2%-1.4% PVA-0588, 0.5%-0.75% PVP-K30, 1-2% Panax notoginseng extract, 1.5%-2.0% glycerol, 20-30% ethanol, 20-30% ethyl acetate, and the balance is water.

[0021] The hemostatic spray film is characterized in that the formula is any one of the following by mass percentage:

[0022] 1.4% PVA-0588, 0.75% PVP-K30, 2% Panax notoginseng extract, 2.0% glycerol, 20% ethanol, 25% ethyl acetate, and the balance is water;

[0023] 1.2% PVA-0588, 0.25% PVP-K30, 2% Panax notoginseng extract, 2.0% glycerol, 20% ethanol, 25% ethyl acetate, and the balance is water;

[0024] 1.4% PVA-0588, 0.5% PVP-K30, 2% Panax notoginseng extract, 2.0% glycerol, 20% ethanol, 30% ethyl acetate, and the balance is water;

[0025] 1.4% PVA-0588, 0.5% PVP-K30, 2% Panax notoginseng extract, 2.0% glycerol, 20% ethanol, 20% ethyl acetate, and the balance is water;

[0026] 1.4% PVA-0588, 0.5% PVP-K30, 2% Panax notoginseng extract, 2.0% glycerol, 25% ethanol, 25% ethyl acetate, and the balance is water;

[0027] 1.6% PVA-0588, 0.25% PVP-K30, 2% Panax notoginseng extract, 2.0% glycerol, 25% ethanol, 25% ethyl acetate, and the balance is water;

[0028] 1.6% PVA-0588, 0.5% PVP-K30, 2% Panax notoginseng extract, 2.0% glycerol, 20% ethanol, 25% ethyl acetate, and the balance is water;

[0029] 1.2% PVA-0588, 0.5% PVP-K30, 2% Panax notoginseng extract, 2.0% glycerol, 25% ethanol, 20% ethyl acetate, and the balance is water;

[0030] 1.3% PVA-0588, 0.75% PVP-K30, 2% Panax notoginseng extract, 2.0% glycerol, 20% ethanol, 23% ethyl acetate, and the balance is water.

[0031] The preparation method of the hemostatic spray film is characterized in that the formulated amounts of PVA-0588, PVP-K30, Panax notoginseng extract, glycerol, ethanol and ethyl acetate are mixed, and water is added to make up the volume to obtain the spray film.

[0032] The comprehensive scoring method for spray film agents in reference [6] sets evaluation indicators. The specific scoring criteria are as follows:

[0033] (1) The full score for spraying is 25 points. Spray at a distance of 15 cm from the white cardboard and observe the spray effect at the nozzle. Mist spray is the best, with a score of 21-25. Semi-mist spray is the second best, with a score of 15-20. Dispersed linear spray is the third best, with a score of 10-15. Spraying in a few sparse lines is the worst, with a score of 1-10.

[0034] (2) The full score for film-forming time is 25 points. Take an appropriate amount of film-forming agent solution, evenly apply it on a glass slide, place it in a 37°C oven, and record the film-forming time. If the film-forming time is less than or equal to 9 minutes, it is considered full score. For every 30 seconds increase from 9 minutes, the score will be reduced by 1 point from the 25 points.

[0035] (3) The maximum score for the toughness of the membrane is 25 points. If 150 μl of the sample is smeared on a glass slide and can be completely torn off, it is considered to be a full score of 25 points. The score is given according to the ratio of the residual area on the slide after torn off to the total area of ​​the membrane. 0% is the full score. For every 1% increase, 1 point is reduced from 25 points. If the membrane cannot be formed and cannot be torn off, the score is 10 points.

[0036] (4) The full score of the film performance is 25 points, including 5 points for the film maintenance time, 10 points for uniformity and 10 points for thickness. The film maintenance time is judged by the degree to which the film can maintain its integrity, with 8 hours as the full score and a score between 0 and 5 points. The film is scored based on whether there are bubbles and the uniformity of the film surface. If the film is bubble-free, transparent and uniform, the score is between 5 and 10. If the film has bubbles, the score is between 0 and 5. After the film is formed, the film is rolled into strips. If it can be rolled into thin threads, it is the best thickness and is scored between 7 and 10. If a layer of film can be completely torn off without deformation, it is considered too thick and is scored below 7. If it cannot be rolled into thin threads, it is considered too thin and is scored below 7.

[0037] The total score of spray effect, film forming time, film toughness and film performance is added together to get the comprehensive score.

[0038] Beneficial effects

[0039] 1. The innovation of the present invention lies in the proportional relationship between components and contents. The present invention combines Panax notoginseng extract with a spray film, designs and optimizes a method for preparing a rapid hemostatic spray film. Taking Panax notoginseng extract as the main hemostatic component, the content of notoginseng in the extracts of different extraction methods is determined to select the advantageous extraction method; using polyvinyl alcohol and polyvinyl pyrrolidone as the main film-forming materials, a spray film is prepared, and the prescription process is optimized; an evaluation standard for the spray film is established, the properties of the spray film are examined, and the total score of the spray effect, film-forming time, film toughness and film performance is added together to form a comprehensive score. Specifically, the present invention first uses a single-factor investigation experiment for optimization to find the four most influential factors (A: PVA-0588, B: PVP-K30, C: Ethanol, D: Ethyl acetate), but a single-factor investigation cannot obtain accurate results, because the single factor does not take into account the mutual influence between the various factors, resulting in the optimization conditions that may not achieve the purpose of the present invention. Therefore, we further investigated the interaction of four factors (A: PVA-0588, B: PVP-K30, C: Ethanol, D: Ethyl acetate) at a fixed ratio (2% Panax notoginseng extract, 2% glycerol) using a central composite design-response surface methodology. One-step optimization revealed that multiple formulations achieved a comprehensive score of 90 or above, and that these formulations met the objectives of the present invention. As shown in Table 10, the present invention exhibited different effects on spraying, film formation time, film toughness, film performance, duration, uniformity, and thickness under different ratios of the four factors, with no regular pattern. For example, the comprehensive score did not necessarily increase with increasing PVA-0588 content. In fact, the four factors interacted with 2% Panax notoginseng extract and 2% glycerol to form a film. Since specific experiments are required to obtain results before the central composite design-fitting can be carried out, Table 11 shows the best results after fitting through actual experimental effects. Therefore, it is not possible to reasonably predict the results simply through the central composite design software. The central composite design-response surface method for the optimization of the prescription of Panax notoginseng extract spray film is only used for further evaluation, and the test results cannot be predicted using this software alone.

[0040] 2. The preparation method of the present invention allows mixing at room temperature, without the need for high temperatures, ensuring that the notoginseng in the Panax notoginseng extract is not destroyed. Furthermore, the film is stable in the film-forming solvent, with all components fully dissolved without precipitation. The film formulation of the present invention forms a fine mist that is thin, easy to tear, has good toughness, a long film hold time, high transparency, high uniformity, and moderate thickness, with an overall score of 90 or above.

[0041] 3. The present invention achieved an optimal ratio formula, exhibiting excellent adhesion (17.0861g), good moisture retention, good comfort (2.03%), and good breathability. The effect of the spray on the bleeding time of mice after tail amputation: Compared with the blank control group, the Panax notoginseng extract spray group significantly shortened the bleeding time (p<0.05); however, under the larger bleeding volume conditions in our experiment, the bleeding time of the blank spray group was not significantly different from that of the blank control group. This indicates that when blood flow is high, the film formed by the spray alone is not very effective. This is because after the spray is sprayed onto the wound, a film formation process is required. When the blood flow rate exceeds this process, the film will break through the liquid to be formed, resulting in a decrease in hemostatic effect. However, the Panax notoginseng extract spray can achieve a good and rapid hemostatic effect. It is speculated that the Panax notoginseng extract contained in the spray quickly blocks or weakens blood flow, giving the film time to form. The hemostatic activity of the Panax notoginseng extract synergizes with the wound-protective effect of the film, achieving a rapid hemostatic effect.

[0042] 4. From the average content results, it can be seen that water at 60℃ is the best extraction method, and the highest notoginseng content of 3.38w / w% is obtained in the Panax notoginseng extract. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 is the standard curve of notoginseng;

[0044] Figure 2 are two-dimensional contour plots and three-dimensional response surface plots, among which C is the two-dimensional contour plot of CD when factors A and B are fixed; C is the three-dimensional response surface plot of CD when factors A and B are fixed; C is the two-dimensional contour plot of BC when factors A and D are fixed; C is the three-dimensional response surface plot of BC when factors A and D are fixed;

[0045] Figure 3 The figure shows the effect of the spray film on the bleeding time of mice after tail cutting. *p<0.05 indicates a significant difference compared with the blank control group. DETAILED DESCRIPTION

[0046] Example 1

[0047] 1. Operation process of the present invention

[0048] The present invention combines Panax notoginseng extract with a film-spraying agent to design and optimize a method for preparing a rapid hemostatic film-spraying agent. Using Panax notoginseng extract as the primary hemostatic component, the content of notoginseng in the extracts obtained from different extraction methods was determined to select the optimal extraction method. Polyvinyl alcohol and polyvinyl pyrrolidone were used as the primary film-forming materials to create the film-spraying agent, and the formulation process was optimized. Evaluation criteria for the film-spraying agent were established, and the properties of the film-spraying agent were examined. A comprehensive scoring system was used to determine the optimal ratio, where the comprehensive score was calculated by summing the total scores of the spraying effect, film-forming time, film toughness, and film performance.

[0049] A hemostatic spray film comprising a Panax notoginseng extract, a film-forming material, a film-forming solvent, glycerol, and water. To obtain the optimal formulation, the following experiments were conducted:

[0050] 2 Materials and Methods

[0051] 2.1 Experimental Materials and Instruments

[0052] 2.1.1 Reagents

[0053] Ethanol (chromatographically pure, Anhui Tiandi High Purity Solvent Co., Ltd.); ethyl acetate (Nanjing Chemical Reagent Co., Ltd.); methanol (chromatographically pure, Tedia, USA);

[0054] 2.1.2 Instruments

[0055] A magnetic pH meter (PHS-3C, Shanghai Yidian Scientific Instrument Co., Ltd.); an electronic analytical balance (EX125DZH, Ohaus Instruments (Changzhou) Co., Ltd.); an ultrasonic cleaner (BD-5200DTD, Nanjing Beidi Experimental Instrument Co., Ltd.); an ultraviolet lamp analyzer (Betty Shanghai Instrument Co., Ltd.); a high-speed centrifuge (D3024R, Dalong Xingchuang Experimental Instrument (Beijing Co., Ltd.); a thermal constant-temperature heating magnetic stirrer (DF-101S, Henan Yuhua Instrument Co., Ltd.); and an ultrasonic cleaner (BD-5200DTD, Nanjing Beidi Experimental Instrument Co., Ltd.) were used. Laboratory Instrument Co., Ltd.); magnetic stirrer (85-2A, Shanghai Silu Instrument Co., Ltd.); vortex mixer (XW-80A, Shanghai Huxi Analytical Instrument); vacuum rotary evaporator (RE52AA, Shanghai Yarong Biochemical Instrument Factory); circulating water vacuum pump (SHZ-D(III), Nanjing Beidi Laboratory Instrument Co., Ltd.); freeze dryer (LGJ-10A-50, Nanjing Beidi Laboratory Instrument Co., Ltd.); constant temperature oscillator (ZD-85, Changzhou Jintan Jingda Instrument Manufacturing Co., Ltd.); high performance liquid chromatography-ultraviolet (HPLC-UV) (LC20AD-SPD 20A, Shimadzu).

[0056] 2.2.3 Experimental animals

[0057] 24 male ICR mice (license number: SCXK(Su): 2020-0009), 7 weeks old, weighing 20±2 g, certificate number: NO.202223758; laboratory animal practitioner certificate number: 220201305.

[0058] 2.2 Preparation of Panax notoginseng extract

[0059] 2.2.1 Preparation of Panax notoginseng extract

[0060] Weigh 100g of coarse particles of Panax notoginseng medicinal material, soak them in an equal volume of corresponding solvent for 24h, and extract them by ultrasonication of alcohol solution, heating of alcohol solution, ultrasonication of aqueous solution and heating of aqueous solution. Ultrasonication group: ultrasonication for 20min / time, filtration, adding a certain amount of corresponding solvent, repeating the above ultrasonication operation for a total of 3 times (ultrasonic frequency is 40kHz, power is 180W); (2) Heating group: 60℃ water bath, 20min / time, filtration, adding a certain amount of corresponding solvent, repeating the above heating operation for a total of 3 times. Combine the subsequent filtrates. The aqueous extract is directly freeze-dried. The alcohol extract is rotary evaporated in a 60℃ water bath until about 30% remains, and then freeze-dried. Weigh the freeze-dried product and calculate the yield of Panax notoginseng extract according to the following formula:

[0061]

[0062] Wherein, P is the yield, m is the weight of freeze-dried Panax notoginseng extract, and M is the weight of coarse particles.

[0063] 2.2.2 Methodology for determination of notoginseng content

[0064] 2.2.2.1 Chromatographic conditions

[0065] High performance liquid chromatography (HPLC) analysis was performed using a C18 column (Hanbang Phecda, 250 mm × 4.6 mm, 5 μm) with a flow rate of 1 mL / min, a column temperature of 35 °C, a detection wavelength of 220 nm, and an injection volume of 5 μL.

[0066] 2.2.2.2 Solution preparation

[0067] 2.2.2.2.1 Preparation of reference solution

[0068] Accurately weigh 50 mg of Panax notoginseng reference substance into a 50 ml brown volumetric flask, ultrasonically dissolve and dilute to the mark with water, and shake well to make a 1 mg / ml reference substance stock solution. Accurately measure 5 ml of the stock solution into a 25 ml brown volumetric flask, dilute to the mark with water, and shake well to obtain a 0.2 mg / ml (200 μg / ml) reference substance solution.

[0069] 2.2.2.2.2 Preparation of test solution

[0070] Place 500 mg of dried Panax notoginseng extract in a 50 ml brown volumetric flask, ultrasonically dissolve and dilute to the mark with water, then shake well to prepare a 10 mg / ml reference stock solution. Accurately measure 5 ml of the stock solution into a 25 ml brown volumetric flask, dilute to the mark with water, shake well, and filter through a 0.45 μm microporous membrane to obtain a 2 mg / ml test solution.

[0071] 2.2.2.3 Methodological validation

[0072] 2.2.2.3.1 Standard curve

[0073] Accurately pipette an appropriate amount of the above-mentioned reference solution into a 10 mL brown volumetric flask, add purified water to dilute to the scale, and prepare a series of reference solutions with concentrations of 2, 5, 10, 25, 50, 100, and 200 μg / ml, respectively. Shake well, and pipette 5 μl of each solution and inject it in sequence. Determine the peak area value of Panax notoginseng according to the above-mentioned chromatographic conditions, and perform linear regression on the reference concentration (x) using the peak area integral value (y).

[0074] 2.2.2.3.2 Precision test

[0075] Take the same concentration of reference solution (50 μg / ml), inject it 5 times continuously, and record the peak area.

[0076] 2.2.2.2.3 Repeatability test

[0077] Repeatability test: Take samples from the same batch and follow the method under "Preparation of test solution" to prepare 5 test solutions, and measure them separately.

[0078] 2.2.2.3.4 Stability test

[0079] Accurately pipette the test solution of the same concentration and inject it for determination at 0, 6, 12, 24, and 48 hours.

[0080] 2.2.3 Determination of notoginseng content

[0081] Take the test solution, draw 5 μl of each and inject it in sequence, determine the notoginseng peak area value according to the above chromatographic conditions, and calculate the notoginseng content by the regression curve.

[0082] 2.3 Formulation design and optimization of Panax notoginseng extract spray film

[0083] Polyvinyl alcohol (PVA) is commonly used as a film-forming material due to its low price, rapid film formation, and uniformity. PVA0588 is a commonly used grade. PVA alone often exhibits poor toughness and adhesion, so it is often combined with other materials to enhance the film's properties. Polyvinyl pyrrolidone (PVP), while not particularly strong on its own, can improve the material's toughness and adhesion, leading to the use of PVA and PVP as a blended film-forming material.

[0084] The preferred solvents for film-forming preparations include ethanol, isopropanol, and ethyl acetate. Ethanol and ethyl acetate are the most widely used, dissolving most inorganic and organic compounds, being miscible with a wide range of organic solvents, exhibiting moderate evaporation times, exhibiting minimal toxicity, and demonstrating good patient compliance. Therefore, ethanol and ethyl acetate were used as the mixed solvents for the film-forming spray in this study.

[0085] 2.3.1 Single Factor Investigation

[0086] 2.3.1.1 Evaluation indicators

[0087] The spray formulation was preliminarily evaluated using the spraying condition, adhesion stop flow and film formation time, and film performance as evaluation indicators. The specific evaluation criteria are as follows:

[0088] Spraying conditions: Each prescription solution was sprayed using a spray bottle, and the spray state was observed and divided into four conditions: (1) linear; (2) between linear and mist, tending to mist (fine mist, tending to linear); (3) between linear and mist (fine mist);

[0089] The adhesion stop flow and film formation time were measured by applying an appropriate amount of each prescription solution on a 1 cm × 2 cm area on a glass slide and placing it in a 37°C oven. The stop flow time was recorded. When the surface was completely dry, it was considered to be a film and the film formation time was recorded.

[0090] The film's appearance properties, such as transparency, uniformity, and thickness, were observed, and the film's toughness was evaluated by whether it could be completely torn off and its stretchability after being rolled into strips.

[0091] 2.3.1.2 PVA-0588 dosage investigation

[0092] In the fixed formula, PVP-K30 was 50 mg, Panax notoginseng extract was 0.2 g, glycerol was 0.2 g, ethanol was 2 g, and ethyl acetate was 2 g. 0.08, 0.10, 0.12, 0.14, and 0.16 g of PVA-0588 were used to prepare the spray film, and the volume was adjusted to 10 g with water. 2.3.1.3 PVP-K30 dosage study

[0093] The fixed prescription contained 0.12 g of PVA-0588, 0.2 g of Panax notoginseng extract, 0.2 g of glycerol, 2 g of ethanol, and 2 g of ethyl acetate. 25, 50, 75, 100, and 125 mg of PVP-K30 were used to prepare the spray film, and the volume was adjusted to 10 g with water.

[0094] 2.3.1.4 Investigation of the dosage of Panax notoginseng extract

[0095] In the fixed prescription, PVA-0588 is 0.12 g, PVP-K30 is 50 mg, glycerol is 0.2 g, ethanol is 2 g, and ethyl acetate is 2 g. The spray film is prepared with 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, and 0.35 g of Panax notoginseng extract, respectively, and the volume is adjusted to 10 g with water.

[0096] 2.3.1.5 Investigation of glycerol dosage

[0097] In the fixed formula, PVA-0588 is 0.12g, PVP-K30 is 50mg, Panax notoginseng extract is 0.2g, ethanol is 2g, and ethyl acetate is 2g. 0.10, 0.15, 0.20, 0.25, and 0.30g of glycerol are used to prepare the spray film, and the volume is adjusted to 10g with water. 2.3.1.6 Study on the amount of ethanol

[0098] In the fixed prescription, PVA-0588 is 0.12 g, PVP-K30 is 50 mg, Panax notoginseng extract is 0.2 g, glycerol is 0.2 g, and ethyl acetate is 2 g. The spray film is prepared with 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, and 4.0 g of ethanol, respectively, and the volume is adjusted to 10 g with water.

[0099] 2.3.1.7 Investigation of the dosage of ethyl acetate

[0100] In the fixed prescription, PVA-0588 is 0.12 g, PVP-K30 is 50 mg, Panax notoginseng extract is 0.2 g, glycerol is 0.2 g, and ethanol is 2 g. The spray film is prepared with 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, and 4.0 g of ethyl acetate, respectively, and the volume is adjusted to 10 g with water.

[0101] 2.3.2 Optimization of the formulation of Panax notoginseng extract spray film by central composite design-response surface methodology

[0102] 2.3.2.1 Evaluation indicators

[0103] The comprehensive scoring method for spray film agents in reference [6] sets evaluation indicators. The specific scoring criteria are as follows:

[0104] (1) The full score for spraying is 25 points. Spray at a distance of 15 cm from the white cardboard and observe the spray effect at the nozzle. Mist spray is the best, with a score of 21-25. Semi-mist spray is the second best, with a score of 15-20. Dispersed linear spray is the third best, with a score of 10-15. Spraying in a few sparse lines is the worst, with a score of 1-10.

[0105] (2) The full score for film-forming time is 25 points. Take an appropriate amount of film-forming agent solution, evenly apply it on a glass slide, place it in a 37°C oven, and record the film-forming time. If the film-forming time is less than or equal to 9 minutes, it is considered full score. For every 30 seconds increase from 9 minutes, the score will be reduced by 1 point from the 25 points.

[0106] (3) The maximum score for the toughness of the membrane is 25 points. If 150 μl of the sample is smeared on a glass slide and can be completely torn off, it is considered to be a full score of 25 points. The score is given according to the ratio of the residual area on the slide after torn off to the total area of ​​the membrane. 0% is the full score. For every 1% increase, 1 point is reduced from 25 points. If the membrane cannot be formed and cannot be torn off, the score is 10 points.

[0107] (4) The full score of the film performance is 25 points, including 5 points for the film maintenance time, 10 points for uniformity and 10 points for thickness. The film maintenance time is judged by the degree to which the film can maintain its integrity, with 8 hours as the full score and a score between 0 and 5 points. The film is scored based on whether there are bubbles and the uniformity of the film surface. If the film is bubble-free, transparent and uniform, the score is between 5 and 10. If the film has bubbles, the score is between 0 and 5. After the film is formed, the film is rolled into strips. If it can be rolled into thin threads, it is the best thickness and is scored between 7 and 10. If a layer of film can be completely torn off without deformation, it is considered too thick and is scored below 7. If it cannot be rolled into thin threads, it is considered too thin and is scored below 7.

[0108] The total score of spray effect, film forming time, film toughness and film performance is added together to get the comprehensive score.

[0109] 2.3.2.2 Star Point Design

[0110] Based on the single factor experiment, the four most influential factors were selected as the main influencing factors. The comprehensive score of the spray film was used as the evaluation index to optimize the prescription of the Panax notoginseng extract spray film. The factors and levels are shown in Table 1.

[0111] Table 1 Star point design factor level table

[0112] Table 1 Levels and the experimental conditions for Box-Behnken design

[0113]

[0114] 2.2.3 Best Prescription Verification

[0115] Three batches of spray film containing Panax notoginseng extract were prepared based on the optimal formula obtained by central composite design-response surface methodology and scored.

[0116] 2.4 Evaluation of spraying agents

[0117] The optimal formulation process was optimized by central composite design-response surface methodology to prepare three batches of preparations for film spray evaluation.

[0118] 2.4.1 Adhesion test

[0119] Take a 2cm×4cm piece of pig skin and fix it on a glass slide a (with a hook). Apply the prepared spray agent on another glass slide b (with a hook) and dry it to form a film. Put it in contact with a and apply 100g pressure for 20s. Fix the glass slide a vertically and tie a plastic bag on the hook of b. Slowly add water to the plastic bag until it separates due to excessive tension. Weigh the weight of the water in the plastic bag, which is the tissue adhesion force.

[0120] 2.4.2 Moisture retention test

[0121] Place in two desiccators with relative humidity RH of 45% and 75% respectively, weigh after 4, 8, 12, 16 and 24 hours, and calculate the residual moisture rate Rh: Rh (%) = (H0 / Hn) × 100%, H0 and Hn are the mass of water before and after placement.

[0122] 2.4.3 Comfort test

[0123] The percentage elongation at break is the ratio of the elongated length of the film at the moment of tensile fracture to the initial length of the film, which is used to evaluate the toughness of the wound dressing.

[0124] 2.4.4 Air permeability test

[0125] Take a vial, add an appropriate amount of silica gel, and cover the vial mouth with a film. Accurately weigh each vial and record the weight. Place the prepared test sample in a desiccator filled with silica gel for 12 hours. After removal, accurately weigh and record the weight. Next, place the vial in a desiccator containing a saturated NaCl solution (RH 75%). Accurately weigh the vial at time points 0, 12, 24, and 48 hours, and measure the water vapor permeability of the film.

[0126] 2.5 In vitro hemostatic activity of the spray film

[0127] 2.5.1 Preparation of Panax notoginseng extract hemostatic spray

[0128] A hemostatic spray film containing Panax notoginseng extract was prepared according to the optimal prescription obtained from the star point experiment.

[0129] 2.5.2 Preparation of blank spray film

[0130] According to the best prescription obtained from the star point experiment, a blank spray film was prepared without adding Panax notoginseng extract.

[0131] 2.5.3 Effect of the spray on the bleeding time of mice after tail amputation

[0132] The effect of a hemostatic spray containing Panax notoginseng extract on the bleeding time of surface wounds was determined using the mouse tail amputation method [7,8]. To simulate a high blood flow, the distance between the transverse section and the tip of the mouse tail was increased from the 0.5 cm commonly used in the literature to 3 cm. Twenty-four male ICR mice were randomly divided into three groups, with eight mice in each group. The tails were transversely cut with surgical scissors 3 cm from the tip of the mouse tail. After the first drop of blood flowed out, it was absorbed with filter paper. The bleeding section was then completely placed in the Panax notoginseng extract spray (Panasonic notoginseng extract spray group) or a blank spray (blank spray group), or left untreated (blank control group). The timer was started after the blood flowed out, and the bleeding time was recorded until the blood stopped seeping out. The experimental results of each group were compared.

[0133] 2.6 Statistical analysis

[0134] All data were statistically analyzed by one-way analysis of variance (ANOVA), followed by Tukey's or Dunnett T3 test using SPSS26 software (IBM, Chicago, USA), and p < 0.05 was considered statistically significant.

[0135] 3 Experimental results and discussion

[0136] 3.1 Preparation of Panax notoginseng extract

[0137] 3.1.1 Properties and Yield of Panax notoginseng Extract

[0138] The alcohol-extracted lyophilized product was light brown in color and in the form of loose blocks; the water-extracted lyophilized product was light khaki in color and in the form of a loose film that would break apart upon light touch. The yields obtained by the four extraction methods are shown in Table 1.

[0139] Table 2 Yields of Panax notoginseng extracts using different extraction methods

[0140] Table 2 The yield of Panax notoginseng extract by different extraction methods

[0141] Extraction method Yield Ultrasonic alcohol extraction 24.15％ Heating alcohol extraction 27.05％ Ultrasonic water extraction 28.70％ Heating water extraction 27.10％

[0142] 3.1.2 Methodology for determination of notoginseng content

[0143] 3.1.2.1 Standard curve

[0144] like Figure 1 As shown in Figure 2, the linear relationship of Panax notoginseng was good in the concentration range of 2-200 μg / mL, with r2 = 1. The regression equation was: A = 5730.6C-1070.4, r 2 =1.

[0145] 3.1.2.2 Precision test

[0146] The same concentration of reference solution (50 μg / ml) was taken and injected 5 times continuously, and the peak area was recorded. The result showed that the RSD of the notoginseng peak area was 0.08% (n=5), indicating that the instrument had good precision.

[0147] 3.1.2.3 Repeatability test

[0148] The RSDs of the notoginseng peak areas of the Panax notoginseng extracts obtained by the four extraction methods were: alcohol ultrasonic RSD = 0.31% (n = 5), water ultrasonic RSD = 0.12% (n = 5), alcohol 60°C RSD = 0.26% (n = 5), and water 60°C RSD = 0.31% (n = 5), demonstrating the good reproducibility of this method.

[0149] 3.1.3.4 Stability test

[0150] The RSDs of the notoginseng peak areas of the Panax notoginseng extracts obtained by the four extraction methods were: alcohol ultrasound RSD = 0.24% (n = 5), water ultrasound RSD = 0.19% (n = 5), alcohol 60℃ RSD = 0.26% (n = 5), and water 60℃ RSD = 0.07% (n = 5). The peak area of ​​the test solution was basically stable within 48 hours.

[0151] 3.1.3 Determination of notoginseng content

[0152] Three test solutions were prepared according to the method under "Preparation of test solution", and 5 μl was injected into each sample. The average content (i.e., mass percentage, w / w, %) and RSD (%) of the notoginseng extracts obtained by the four extraction methods were calculated and shown in Table 2.

[0153] Table 3 Notoginseng content in Panax notoginseng extracts using different extraction methods

[0154] Table 3 The Content of Panax notoginseng in extract by different extraction methods

[0155]

[0156] The results of the average content showed that the best extraction method was water at 60℃, which resulted in the highest content of notoginseng in the Panax notoginseng extract. The RSD value of the content showed that the results were reliable.

[0157] 3.2 Formulation design and optimization of Panax notoginseng extract spray film

[0158] 3.2.1 Single-factor observation experiment

[0159] 3.2.1.1 PVA-0588 dosage investigation

[0160] The results of the PVA-0588 dosage study are shown in Table 4. As the PVA-0588 content increases, the spray pattern gradually shifts from a mist to a linear pattern, and the film thickness increases slightly. At lower content levels, the film is too thin and has slightly poor toughness. Considering both the spray and film-forming properties, the optimal PVA-0588 dosage range is 1.2%-1.4%.

[0161] Table 4 Effect of PVA-0588 dosage on film spray formulation

[0162] Table 4 Effect of PVA-0588 dosage on formulation of spray film

[0163] content% Injection situation No flow / min Film forming / min Film formation 0.8 fine mist 3-4 10 The film is thin, transparent, tearable, and has poor toughness 1.0 fine mist 3 10 Thin film, tearable, poor toughness 1.2 fine mist 3 10 The film is thin, transparent, tearable, and slightly tough 1.4 Fine mist, linear 3 10 The film is thin, easy to tear off, and slightly tough 1.6 Linear 4-5 10 The film is thick, easy to tear off, and has good toughness

[0164] 3.2.1.2 Investigation of PVP-K30 dosage

[0165] The results of the PVP-K30 dosage study are shown in Table 5. As the PVP-K30 content increases, the spray pattern gradually shifts from a fine mist to a linear pattern. Film thickness and film-forming time remain largely unchanged, but film toughness gradually increases. Considering both spray and film-forming conditions, the optimal PVP-K30 dosage range is 0.5% to 0.75%.

[0166] Table 5 Effect of PVP-K30 dosage on spray film formulation

[0167] Table 5 Effect of PVP-K30 dosage on formulation of spray film

[0168]

[0169] 3.2.1.3 Investigation of the dosage of Panax notoginseng extract

[0170] The results of the investigation of Panax notoginseng extract dosage are shown in Table 6. Variations in the Panax notoginseng extract content had little effect on the film's spray characteristics, resulting in a mostly fine mist, thin film, and good toughness. Because the Panax notoginseng extract used is a powder, excessive levels of Panax notoginseng extract lead to incomplete dissolution and uneven solution, resulting in different spray characteristics and film formation, with a more linear spray pattern and thicker films with poor toughness. Considering the spray characteristics, film formation, and the efficacy of Panax notoginseng extract, the optimal dosage of Panax notoginseng extract is 2%.

[0171] Table 6 Effect of the dosage of Panax notoginseng extract on the spray film formulation

[0172] Table 6 Effect of Triax notoginseng extract dosage on formulation ofspray film

[0173]

[0174] 3.2.1.4 Investigation of glycerol dosage

[0175] The results of the glycerol dosage study are shown in Table 7. Increasing the glycerol dosage resulted in a more linear spray pattern, increased film toughness, and easier film removal, with minimal impact on film formation time. Considering both spray and film formation, the glycerol dosage range was 1.5% to 3.0%, with 2% being the optimal level.

[0176] Table 7 Effect of glycerol dosage on spray film formulation

[0177] Table 7 Effect of glycerol dosage on formulation of spray film

[0178]

[0179]

[0180] 3.2.1.5 Investigation of ethanol dosage

[0181] The results of the ethanol dosage investigation are shown in Table 8. When the ethanol content is low, the solution stratifies, exhibits poor stability, and exhibits uneven mixing, which affects film formation. When ethanol is used as a volatile organic solvent, increasing the ethanol dosage can shorten film formation time, facilitating rapid film formation on human skin. Increasing the ethanol content significantly reduces the spraying effect, while the film toughness gradually deteriorates. At 40% ethanol, solution stability deteriorates, with stratification. Considering both spraying and film formation, the ethanol dosage should be between 20% and 35%.

[0182] Table 8 Effect of ethanol dosage on spray film formulation

[0183] Table 8 Effect of ethanol dosage on formulation of spray film

[0184]

[0185] 3.2.1.6 Investigation of the dosage of ethyl acetate

[0186] The results of the investigation on the dosage of ethyl acetate are shown in Table 9. When the content of ethyl acetate is 10%-20%, the spray condition is fine mist, the solution is uniform, the film formed is thin, easy to tear off, and has good toughness. When the dosage of ethyl acetate exceeds 25%, the stability of the solution decreases, and there is stratification, which may be related to the solubility of the film-forming material. Considering the comprehensive spraying and film-forming conditions, the dosage of ethyl acetate is 10-20%. However, since it is a fixed factor, the current optimal dosage is 10-20%. Considering the possible impact of ethanol, subsequent experiments use ethyl acetate at 20-30% for investigation, that is, stratification occurs at the current ratio, but stratification may not occur for different ethanol solutions). Ethyl acetate requires comprehensive consideration of other factors. A single factor cannot fully meet the screening requirements.

[0187] Table 9 Effect of ethyl acetate dosage on spray film formulation

[0188] Table 9 Effect of ethyl acetate dosage on formulation of spray film

[0189]

[0190] The above single-factor analysis identified four significant factors (A: PVA-0588, B: PVP-K30, C: Ethanol, and D: Ethyl acetate). However, single-factor analysis cannot yield accurate results because it fails to consider the interactions between these factors. Consequently, the optimized conditions may not achieve the objectives of the present invention. Therefore, a central composite design coupled with response surface methodology was employed for a one-step optimization, considering the impact of each factor on the overall effect (comprehensive score).

[0191] 3.2.2 Optimization of the formulation of Panax notoginseng extract spray film by central composite design-response surface methodology

[0192] 3.2.2.1 Star point design results

[0193] Based on the results of the single-factor test, the comprehensive score was used as the response value. According to the Box-Behnken design factor level table, the experimental plan was designed using the software Design-Expert 13. A fixed amount of (2% Panax notoginseng extract, 2% glycerol) was used to design the experimental plan and the experiment was conducted according to the single-factor investigation experimental method in 3.2.1. The results are shown in Table 10.

[0194] Table 10 Star point experiment design table and results

[0195] Table 10 Box-Behnken design and results

[0196]

[0197]

[0198] 3.2.2.2 Model Fitting

[0199] The effect of the prescription ingredient ratios on the scores is shown in Table 11. The model's F-value of 4.67 indicates that the model is significant. Only 0.34% model noise results in such a strong F-value. The underfit F-value of 3.43 indicates that the underfit is not significant relative to the pure error.

[0200] Table 11 Star point design experiment scores

[0201] Table 11 Comprehensive score of Box-Behnken factorial design

[0202]

[0203]

[0204] The two-dimensional contour plot and three-dimensional response surface plot of the model are shown in Figure 2 The resulting polynomial equation is as follows: Comprehensive score = -472.183 + 452.417A + 238.933B + 9.86667C + 8.68333D - 95AB + 0.25AC - 2.23546e-13AD - 4.6BC + 2.4BD - 0.11CD - 150.417A 2 -64.2667B 2 -0.135667C 2 -0.165667D 2 .

[0205] The software recommends the optimal process as follows: A. PVA 0588 1.31%, B. PVP-K30 0.744%, C. Ethanol 20.18%, D. Ethyl acetate 23.368%. Considering actual production practices and to ensure simplicity and feasibility, the optimal process is determined to be A. PVA 0588 1.3%, B. PVP-K30 0.75%, C. Ethanol 20%, and D. Ethyl acetate 23%.

[0206] As can be seen from the above, the comprehensive score is above 90 points, and the film formation meets the requirements. As shown in Table 10, the present invention has different effects on spraying, film formation time, film toughness, film performance, maintenance time, uniformity, and thickness under different proportions of the four factors. There is no regular pattern to be found. For example, the comprehensive score does not necessarily increase with increasing PVA-0588 content. In fact, the four factors interact with 2% Panax notoginseng extract and 2% glycerol to form the film. It is not possible to reasonably predict the results simply through star point design software and need to be verified through experiments.

[0207] Table 11 is the best result after fitting the experimental effect. The premise must be the experimental results. However, due to the different proportions of the experimental effects, the software cannot be reasonably expected. The star point design-response surface method for the optimization of the prescription of Panax notoginseng extract spray film is only used for further evaluation, and the software cannot be used alone to predict the experimental results.

[0208] 3.2.3 Optimal Prescription Verification

[0209] Three batches of Panax notoginseng extract spray films prepared with the optimal prescription were scored, and the results are shown in Table 12. The actual comprehensive score values ​​were similar to the predicted comprehensive score values, indicating that the determined prescription conditions were reliable and feasible.

[0210] Table 12 Best prescription verification

[0211] Table 12 Selected formulation verification test

[0212] Serial number Comprehensive score rating average Predicted value 1 91 2 90 91 91.2 3 92

[0213] 3.3 Evaluation of spraying agents

[0214] 3.3.1 Adhesion test

[0215] The adhesion test results are shown in Table 12.

[0216] Table 12 Adhesion test results

[0217] Table 12 The result of adhesion test

[0218]

[0219] 3.3.2 Moisturizing test

[0220] The results of the moisturizing test at humidity RH = 45% and 75% are shown in Table 13. It has good moisturizing properties.

[0221] Table 13 Moisture Retention Test Results

[0222] Table 13 The result of moisture retention test

[0223]

[0224]

[0225] 3.3.3 Comfort test

[0226] The comfort test results are shown in Table 14.

[0227] Table 14 Comfort test results

[0228] Table 14 The result of comfort degree test

[0229]

[0230] 3.3.4 Air permeability test

[0231] The results of the air permeability test are shown in Table 15.

[0232] Table 15 Air permeability test results

[0233] Table 15 The result of air permeability test

[0234]

[0235] 3.4 Effect of the spray on the bleeding time of mice after tail amputation

[0236] In the mouse tail amputation experiment, the effects of the Panax notoginseng extract spray film group, blank spray film group and blank control group on the bleeding time of the mouse tail were as follows: Figure 3 SPSS 26 software (IBM, Chicago, USA) was used to perform Tukey's test (for homogeneous variance) or Dunnett T3 test (for unequal variance). The results of the homogeneous variance test showed that the data had homogeneous variance. Therefore, significant differences were determined by Tukey's test, and p < 0.05 was considered statistically significant.

[0237] Compared with the blank control group, the Panax notoginseng extract spray film group can significantly shorten the bleeding time (p<0.05); while the blank spray film group had no significant difference in bleeding time compared with the blank control group under the condition of large bleeding volume in our experiment. This shows that when the blood flow is large, the film formed by the spray film alone is not very effective, because after the spray film is sprayed onto the wound, a film-forming process is required. When the blood flow rate exceeds this process, it will break through the liquid surface to be formed into the film, resulting in a decrease in the hemostatic effect. The Panax notoginseng extract spray film can play a good and rapid hemostatic role. It is speculated that the Panax notoginseng extract contained in the spray film plays a role in quickly blocking or weakening the blood flow, giving time for the film to form. The hemostatic activity of the Panax notoginseng extract and the role of the film in protecting the wound synergize to play a rapid hemostatic role.

[0238] References

[0239] [1]Xie H, Chen X, Shen

[0240] [2] Xu Hang, Zhang Lingna, Zhang Xueting, Lin Jiao, Yang Xuehan, Song Hongtao. Research progress of film-forming materials for liquid bandages. Journal of Pharmaceutical Practice 2021;39:299–304.

[0241] [3] Liu Haixia, Zhang Aijun, Zhang Lin. Research progress of liquid bandage. Chinese Journal of Medical Science, 2016: 28–31.

[0242] [4] Chinese Pharmacopoeia Committee. Chinese Pharmacopoeia. China Medical Science and Technology Press; 2020.

[0243] [5] Wang Yanyan. Study on the basic characterization of the effective substances of Panax notoginseng in promoting blood circulation and stopping bleeding. Master. Beijing University of Chinese Medicine, 2006.

[0244] [6] Wang Qian. Development of Pulsatilla Spray Film. Master. Qingdao University of Science and Technology, 2019.

[0245] [7] Yan Tao, Guo Lin, Miao Mingsan. Experimental study on the hemostatic effect of aloe vera. Henan Journal of Traditional Chinese Medicine 2014;34:1680–1. https: / / doi.org / 10.16367 / j.issn.1003-5028.2014.09.088.

[0246] [8] Cheng Qinyuan. Study on the hemostatic activity of chitosan and its biosafety evaluation. Ph.D. Jiangnan University, 2013.

Claims

1. A hemostatic spray film, characterized in that: By mass percentage, the formula is any of the following: 1.4% PVA-0588, 0.75% PVP-K30, 2% Panax notoginseng extract, 2.0% glycerol, 20% ethanol, 25% ethyl acetate, and the balance is water; 1.4% PVA-0588, 0.5% PVP-K30, 2% Panax notoginseng extract, 2.0% glycerol, 20% ethanol, 30% ethyl acetate, and the balance is water; 1.4% PVA-0588, 0.5% PVP-K30, 2% Panax notoginseng extract, 2.0% glycerol, 20% ethanol, 20% ethyl acetate, and the balance is water; 1.4% PVA-0588, 0.5% PVP-K30, 2% Panax notoginseng extract, 2.0% glycerol, 25% ethanol, 25% ethyl acetate, and the balance is water; 1.6% PVA-0588, 0.25% PVP-K30, 2% Panax notoginseng extract, 2.0% glycerol, 25% ethanol, 25% ethyl acetate, and the balance is water; 1.6% PVA-0588, 0.5% PVP-K30, 2% Panax notoginseng extract, 2.0% glycerol, 20% ethanol, 25% ethyl acetate, and the balance is water; 1.2% PVA-0588, 0.5% PVP-K30, 2% Panax notoginseng extract, 2.0% glycerol, 25% ethanol, 20% ethyl acetate, and the balance is water; 1.3% PVA-0588, 0.75% PVP-K30, 2% Panax notoginseng extract, 2.0% glycerol, 20% ethanol, 23% ethyl acetate, and the balance is water; The Panax notoginseng extract is prepared by the following steps: weighing 100 g of coarse Panax notoginseng medicinal material particles, soaking them in an equal volume of corresponding solvent for 24 hours, extracting them by ultrasonication of an alcohol solution, heating an alcohol solution, ultrasonication of an aqueous solution or heating an aqueous solution, filtering, and combining the filtrates; freeze-drying the aqueous extract, and rotary evaporating the alcohol extract in a 60°C water bath until about 30% remains, and freeze-drying; wherein the ultrasonic conditions are as follows: ultrasonic frequency of 40 kHz, power of 180 W, ultrasonication for 20 minutes / time, filtration, and addition of more than twice the volume of solvent for ultrasonic extraction; heating conditions are as follows: 60°C water bath for 20 minutes / time, filtration, addition of more than twice the volume of solvent, and heating extraction.

2. The method for preparing a hemostatic spray film according to claim 1, characterized in that: PVA-0588, PVP-K30, Panax notoginseng extract, glycerol, ethanol and ethyl acetate in a formulated amount were mixed, and water was added to make up the volume to obtain a spray film.

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