A soluble microneedle of a four-yellow composition and its application

Abstract

The present invention provides a four-yellow composition soluble microneedle and its application, relating to the field of transdermal drug delivery technology. A four-yellow composition soluble microneedle is prepared by the following preparation method: an aqueous solution of polyvinyl pyrrolidone, sodium carboxymethyl cellulose and DES four-yellow extract is mixed and centrifuged to obtain a mother liquor; a 3-7wt% hyaluronic acid DES four-yellow extract aqueous solution is configured as a base liquid; the mother liquor is taken to a microneedle mold, vacuumed and dried after ultrasonication to obtain a first layer; the mother liquor is drawn on the first layer for a second time, ultrasonicated and dried to obtain a second layer; the base liquid is taken on the second layer, ultrasonicated, dried, and demoulded to obtain a four-yellow composition soluble microneedle. The four-yellow composition soluble microneedle prepared by the present invention can break through the "obstruction" of the skin stratum corneum, so that the active substance reaches the skin dermis, and efficiently improves the efficacy of the DES four-yellow extract raw material in inhibiting acne, removing acne and soothing the skin.

Description

Technical Field

[0001] The present invention relates to the technical field of transdermal drug delivery, and in particular to a soluble microneedle of a four-yellow composition and applications thereof. Background Art

[0002] Flavonoids in traditional Chinese medicines, such as Scutellaria baicalensis, Phellodendron chinense, Coptis chinensis, and Rhubarb, all possess heat-clearing and detoxifying properties, anti-inflammatory and antibacterial properties, and antioxidant properties. They also have acne-inducing and anti-acne effects, as well as skin repair and soothing properties. Zhang Zhongjing's "Golden Chamber" (Jin Kui Yao Lue) from the Han Dynasty states that the "Sanhuang Xiexin Decoction," composed of Coptis chinensis, Scutellaria baicalensis, and Rhubarb, is a classic formula for purging heat and detoxifying, treating symptoms such as internal heat and internal accumulation of stagnant heat. Furthermore, the Huanglian Jiedu Decoction, originating from the "Emergency Prescriptions for the Elbow," uses "3 liang of Coptis chinensis, 2 liang each of Phellodendron chinense and Scutellaria baicalensis, 14 Gardenia jasminoides, 6 liters of water, decoct to 2 liters, and divide into portions." It functions as a heat-purging and detoxifying agent, specifically treating all forms of heat and fire toxins, and is highly effective in the surgical treatment of carbuncles and furuncles. Based on this ancient recipe, the "Sihuang" extract, combining Phellodendron chinense, Coptis chinensis, Rhubarb, and Scutellaria baicalensis in a specific ratio, not only enhances acne-removing efficacy but also represents a unique and innovative fusion of Chinese herbal medicine and cosmetics.

[0003] The skin is primarily composed of three layers: the outermost stratum corneum, the middle epidermis, and the thickest dermis. The skin is the primary barrier to topical drug delivery, and the stratum corneum acts as a "wall," allowing only certain molecules, such as lipophilic and low-molecular-weight drugs, to pass through. This relatively low permeability presents numerous challenges in the design of topical formulations. The most commonly used transdermal drug delivery methods are subcutaneous injections and topical ointments. These methods are plagued by pain from the needle, poor patient acceptance of the needle, and the difficulty of topical creams in penetrating the stratum corneum, resulting in low bioavailability.

[0004] In recent years, researchers have investigated various topical or transdermal drug delivery systems to improve drug penetration through the skin, such as nanocarrier-loaded topical creams, transdermal patches, and microneedles. Microneedles are needle-like structures with a diameter of less than tens of micrometers and a length of 25-2000 μm. Microneedle transdermal drug delivery involves using an array of microneedles to pierce the stratum corneum, creating micron-scale channels that allow drug molecules or even drug particles to pass directly into the subcutaneous layer, thereby achieving drug delivery and effectively improving the utilization of active substances. Microneedles are categorized into solid microneedles, hollow microneedles, coated microneedles, soluble microneedles, and hydrogel microneedles.

[0005] Dissolving microneedles are made by combining biodegradable polymer materials (such as hyaluronic acid and polyvinylpyrrolidone) with drugs. After piercing the skin, the biodegradable needle gradually degrades in the microenvironment, releasing the drug simultaneously. The drug molecules then penetrate the stratum corneum and are absorbed into the subcutaneous tissue. Dissolving microneedles eliminate the need for removal after drug delivery, as is the case with other microneedles. This significantly improves patient compliance, while also reducing the risk of cross-infection by eliminating the need for reuse.

[0006] Based on this, how to combine Chinese herbal medicine extracts with soluble microneedles to solve the problems of skin delivery and transdermal absorption is an urgent problem to be solved by researchers in this field. Summary of the Invention

[0007] To address these issues, the present invention provides an optimized formula and preparation method for a soluble microneedle formulation of a four-yellow compound, using polyvinyl pyrrolidone (PVP), sodium carboxymethyl cellulose (CMC-Na), hyaluronic acid (HA), and DES four-yellow extract as raw materials. Furthermore, the soluble microneedles of the four-yellow compound are able to penetrate the "obstruction" of the skin's stratum corneum, allowing the active ingredients to reach the dermis, effectively enhancing the effectiveness of the four-yellow compound in inhibiting acne, removing acne, and soothing the skin.

[0008] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0009] In one aspect, the present invention provides a soluble microneedle of a four-yellow composition, which is prepared by the following preparation method:

[0010] 1) mixing polyvinyl pyrrolidone, sodium carboxymethyl cellulose and an aqueous solution of DES four yellow extract, and centrifuging to obtain a mother liquor;

[0011] 2) preparing a 3-7 wt % aqueous solution of hyaluronic acid DES four yellow extract as a base solution;

[0012] 3) applying the mother liquid to the microneedle mold, sonicating, vacuumizing, and drying to obtain the first layer; aspirating the mother liquid on the first layer for the second time, sonicating, and drying to obtain the second layer; applying the base liquid on the second layer, sonicating, drying, and demolding to obtain the soluble microneedles of the four-yellow composition.

[0013] Preferably, in step 1), based on weight, 10-20 parts of polyvinyl pyrrolidone, 1-3 parts of sodium carboxymethyl cellulose, and 75-90 parts of DES four yellow extract are used;

[0014] Further preferably, in step 1), based on weight parts, polyvinyl pyrrolidone 10-15 parts, sodium carboxymethyl cellulose 1-2 parts, DES four yellow extract 75-85 parts;

[0015] More preferably, in step 1), based on weight, 15 parts of polyvinyl pyrrolidone, 2 parts of sodium carboxymethyl cellulose, and 83 parts of DES four yellow extract;

[0016] Preferably, in step 1), the concentration of the aqueous solution of the DES four yellow extract is 20-100 mg / mL. More preferably, in step 1), the concentration of the aqueous solution of the DES four yellow extract is 50 mg / mL.

[0017] Preferably, in step 1), the mixing is stirring for 0.5-1.5 h.

[0018] Preferably, in step 1), the centrifugal speed is 5500-6500 r / min, and the time is 5-15 min; further preferably, in step 1), the centrifugal speed is 5500-6000 r / min, and the time is 5-10 min.

[0019] Preferably, in step 1), the preparation method of the DES four yellow extract comprises the following steps:

[0020] S1: Prepare a deep eutectic solvent: use amino acid as HBA and alcohol or carboxylic acid as HBD, heat and stir to obtain DES;

[0021] S2: preparing DES aqueous solution: mixing the DES prepared in S1 with water to obtain a DES aqueous solution;

[0022] S3: Extraction: Take Phellodendron amurense, Coptis chinensis, Rhubarb and Scutellaria baicalensis, grind and sieve, add DES aqueous solution prepared in S2, ultrasonicate at 55-65℃ for 30-60min, cool to room temperature, centrifuge, take the supernatant and dry to obtain DES four-yellow extract.

[0023] Preferably, in S1, the amino acid is selected from at least one of lysine and arginine; further preferably, in S1, the amino acid is selected from lysine or arginine.

[0024] Preferably, in S1, the alcohol is selected from at least one of xylitol and 1,3-propylene glycol; further preferably, in S1, the alcohol is selected from at least one of xylitol and 1,3-propylene glycol.

[0025] Preferably, in S1, the molar ratio of the HBA to the HBD is 1:2 to 4. Further preferably, in S1, the molar ratio of the HBA to the HBD is 1:2 or 1:4.

[0026] Preferably, in S1, the heating and stirring is stirring at 65-75°C.

[0027] Preferably, in S2, the water is selected from at least one of ultrapure water, deionized water, and distilled water; further preferably, in S2, the water is selected from ultrapure water.

[0028] Preferably, in S2, the mass of the water is 35%-65% of the total mass of the DES aqueous solution. Further preferably, in S2, the mass of the water is 40%-60% of the total mass of the DES aqueous solution. More preferably, in S2, the mass of the water is 40%-50% of the total mass of the DES aqueous solution.

[0029] Preferably, in S2, the mixing is performed by stirring at 55-65°C.

[0030] Preferably, in S3, the DES aqueous solution needs to be cooled to room temperature before transfer and stored away from light.

[0031] Preferably, in S3, the mesh number of the sieve is 60-80 mesh; further preferably, in S3, the mesh number of the sieve is 70 mesh.

[0032] Preferably, in S3, the mass ratio of Phellodendron chinense, Coptis chinensis, Rhubarb and Scutellaria baicalensis is 1-2:1-2:1-2:1-2; further preferably, in S3, the mass ratio of Phellodendron chinense, Coptis chinensis, Rhubarb and Scutellaria baicalensis is 1:1:1:1-2; more preferably, in S3, the mass ratio of Phellodendron chinense, Coptis chinensis, Rhubarb and Scutellaria baicalensis is 1:1:1:2.

[0033] Preferably, in S3, the solid-liquid ratio of Phellodendron chinense, Coptis chinensis, Rhubarb and Scutellaria baicalensis to the DES aqueous solution is 1:20-40; further preferably, in S3, the solid-liquid ratio of Phellodendron chinense, Coptis chinensis, Rhubarb and Scutellaria baicalensis to the DES aqueous solution is 1:20 or 1:40.

[0034] Preferably, in S3, the temperature of the ultrasound is 55-60°C and the time is 30-40 minutes.

[0035] Preferably, in S3, the centrifugal speed is 5500-6500 r / min, and the time is 15-25 min; further preferably, in S3, the centrifugal speed is 5500-6000 r / min, and the time is 15-20 min.

[0036] Preferably, in S3, the specific parameters of the drying are: temperature of 55-65°C, vacuum degree of -0.3 to -1 MPa, and time of 10-14 hours. Further preferably, in S3, the specific parameters of the drying are: temperature of 55-60°C, vacuum degree of -0.5 to -1 MPa, and time of 10-12 hours.

[0037] Preferably, in step 2), the specific operation of the preparation is: mixing HA and the aqueous solution of DES four yellow extract for 0.5-1.5 hours, centrifuging, and removing bubbles to obtain the base solution.

[0038] Preferably, the mixing is stirring.

[0039] Preferably, the centrifugal speed is 5500-6500 r / min, and the time is 5-15 min; further preferably, the centrifugal speed is 5500-6000 r / min, and the time is 5-10 min.

[0040] Preferably, in step 3), the duration of all the ultrasounds is 5-15 min. More preferably, in step 3), the duration of all the ultrasounds is 10 min.

[0041] Preferably, in step 3), the drying temperature is 40-50° C. and the drying time is 1.5-2.5 h; further preferably, in step 3), the drying temperature is 40-45° C. and the drying time is 1.5-2 h.

[0042] Preferably, in step 3), the vacuum degree is -0.3 to -1 MPa, and the time is 5 to 15 min; further preferably, in step 3), the vacuum degree is -0.5 to -1 MPa, and the time is 5 to 10 min.

[0043] In another aspect, the present invention provides the use of the soluble microneedles of the above-mentioned four-yellow composition in the preparation of acne-removing products.

[0044] Compared with the prior art, the present invention has the following beneficial effects:

[0045] The soluble microneedles of the four-yellow compound of the present invention have a simple preparation process, rapid dissolution, and excellent transdermal penetration. Furthermore, the four-yellow compound soluble microneedles, obtained by loading the DES four-yellow extract into the soluble microneedles, not only address the problem of traditional smear-type ointments, where the active ingredients remain on the skin surface and have difficulty penetrating deeper into the skin, but also enable the active ingredients to reach the dermis, effectively enhancing the effectiveness of DES four-yellow in inhibiting acne, removing acne, and soothing the skin, making them of great significance in the fields of transdermal absorption and cosmetics. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 Insert the HE staining picture of ex vivo pig skin for comparative example 2.

[0047] Figure 2 This is the dissolution diagram of the fluorescent soluble microneedles prepared in Comparative Example 1.

[0048] Figure 3Microscope images of the soluble microneedles of the Sihuang composition prepared in Examples 6-8.

[0049] Figure 4 Graphs showing the mechanical properties of the soluble microneedles of the four-yellow composition and blank microneedles prepared in Examples 6-8 and Comparative Example 2.

[0050] Figure 5 This is a full wavelength scanning diagram of the DES four yellow extract prepared in Example 5.

[0051] Figure 6 This is a diagram of the drug loading of the soluble microneedles of the Sihuang composition prepared in Examples 6-8.

[0052] Figure 7 This is a graph of the cumulative transdermal release of the soluble microneedles of the Sihuang composition prepared in Example 7 and the DES Sihuang extract prepared in Example 5.

[0053] Figure 8 This is a skin retention diagram of the soluble microneedles of the Sihuang composition prepared in Example 7 and the DES Sihuang extract prepared in Example 5.

[0054] Figure 9 This is a comparison chart of the retention amounts of the soluble microneedles of the Sihuang composition prepared in Example 7 and the DES Sihuang extract prepared in Example 5.

[0055] Figure 10 This is a plate-coating diagram of different concentrations of the soluble microneedles of the Sihuang composition prepared in Example 7.

[0056] Figure 11 This is an electron micrograph of the soluble microneedles of the Sihuang composition prepared in Example 7. (Electron microscopy conditions: magnification: X10000, accelerating voltage: 15 kV, working distance: 7.8 mm, scale: 1 μm). DETAILED DESCRIPTION

[0057] In order to make the technical means, creative features, purpose and effect of the present invention easy to understand, the present invention is further illustrated below in conjunction with specific embodiment, but the following embodiment is only a preferred embodiment of the present invention, not all. Based on the embodiment in the embodiment, other embodiments obtained by those skilled in the art without making creative work all fall within the protection scope of the present invention. It is worth noting that the raw materials used in the present invention are all common commercial products, and their source is not specifically limited. The technology and scientific terms used in the embodiment have the meaning commonly understood by those of ordinary skill in the art to which the present invention belongs.

[0058] The microneedle mold was purchased from Suzhou Yongqinquan Intelligent Equipment Co., Ltd. Except for the microneedle mold, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0059] Example 1:

[0060] A DES scutellaria baicalensis extract (lysine:citric acid molar ratio of 1:4, water content 50%)

[0061] Weigh 0.1008g of lysine and 0.5251g of citric acid into a 10mL centrifuge tube, heat in a 70℃ water bath and stir at 700-900rpm until a clear and stable DES is obtained. Weigh 0.6272g of ultrapure water into the above centrifuge tube, stir in a 60℃ water bath at 700-900rpm until a transparent and uniform DES aqueous solution is formed, then weigh 0.0625g of Scutellaria baicalensis powder into the above DES aqueous solution (solid-liquid ratio 1:20) and shake evenly. Ultrasonic extraction for 40 minutes at 60℃. After the DES solution cools to room temperature, centrifuge at 6000r / min for 20 minutes, and take the supernatant, which is the corresponding Scutellaria baicalensis DES extract. Vacuum it for 12 hours to remove moisture, which is the DES Scutellaria baicalensis extract.

[0062] Example 2:

[0063] A DES Phellodendron amurense extract (lysine:xylitol molar ratio of 1:2, water content 50%)

[0064] Weigh 0.1005g of lysine and 0.2081g of xylitol into a 10mL centrifuge tube. Heat and stir in a 70°C waterbath at 700-900 rpm until a clear, stable DES solution is obtained. Weigh 0.3483g of ultrapure water into the same centrifuge tube and stir in a 60°C waterbath at 700-900 rpm until a transparent, uniform DES aqueous solution is formed. Then, weigh 0.0328g of Phellodendron amurense powder into the DES aqueous solution (solid-to-liquid ratio 1:20) and shake until evenly combined. Subsequent steps are the same as in Example 1. This is the DES Phellodendron amurense extract.

[0065] Example 3:

[0066] A DES coptis root extract (arginine:malic acid molar ratio of 1:4, water content 40%)

[0067] Weigh 0.1002g of arginine and 0.3078g of malic acid into a 10mL centrifuge tube. Heat and stir in a 70°C waterbath at 700-900 rpm until a clear, stable DES solution is obtained. Weigh 0.6115g of ultrapure water into the same centrifuge tube. Stir in a 60°C waterbath at 700-900 rpm until a transparent, uniform DES solution is formed. Then, weigh 0.0509g of Coptis chinensis powder into the DES solution (solid-to-liquid ratio 1:20) and shake until evenly combined. Subsequent steps are the same as in Example 1. This is the DES Coptis chinensis extract.

[0068] Example 4:

[0069] A DES rhubarb extract (arginine:1,3-propylene glycol molar ratio of 1:2, water content 50%)

[0070] Weigh 0.1006g of arginine and 0.0874g of 1,3-propylene glycol into a 10mL centrifuge tube. Heat and stir in a 70°C water bath at 700-900 rpm until a clear, stable DES solution is obtained. Weigh 0.1922g of ultrapure water into the same centrifuge tube and stir in a 60°C water bath at 700-900 rpm until a transparent, uniform DES aqueous solution is formed. Then, weigh 0.0191g of rhubarb powder into the DES aqueous solution (solid-to-liquid ratio 1:40) and shake until evenly combined. Follow the same steps as in Example 1. This is the DES rhubarb extract.

[0071] Example 5:

[0072] DES Four-Huang Extract, a compound of Phellodendron chinense, Coptis chinensis, Rhubarb and Scutellaria baicalensis

[0073] Weigh 0.5g of DES Phellodendron chinense extract, 0.5g of DES Coptidis rhizome extract, 0.5g of DES Rhubarb extract, and 1g of DES Scutellaria baicalensis extract into a beaker, dissolve in ultrapure water, and transfer to a 25mL volumetric flask to obtain 100mg / mL (compound weight ratio of 1:1:1:2) of DES Sihuang extract.

[0074] Next, 5 mL and 12.5 mL of the 100 mg / mL (compound weight ratio 1:1:1:2) DES four yellow extract obtained in Example 5 were taken out and diluted to a 25 mL volumetric flask with ultrapure water to obtain 20 mg / mL and 50 mg / mL DES four yellow extracts.

[0075] Example 6:

[0076] A soluble microneedle of a four-yellow composition:

[0077] 1. Weigh 1.5 g PVP, 0.2 g CMC-Na, and 8.3 g of the 100 mg / mL DES four-yellow extract obtained in Example 5 into a beaker, stir for 1 hour, and centrifuge at 6000 rpm for 10 minutes to obtain a mother liquor;

[0078] 2. Weigh 0.5 g of HA into another beaker, stir for 1 hour, and centrifuge at 6000 rpm for 10 minutes to prepare the base solution;

[0079] 3. Use a pipette to draw an appropriate amount of mother liquor onto the microneedle mold, ultrasonicate for 10 minutes, and then place it in a vacuum drying oven (vacuum degree: -0.5Mpa) for 10 minutes. After the vacuum is over, ultrasonicate to remove bubbles, and place it in a 45°C drying oven for 2 hours to obtain the first layer. After drying for 2 hours, draw an appropriate amount of mother liquor onto the first layer of the mold again, ultrasonicate for 10 minutes, and then place it in a 45°C drying oven for 4 hours to obtain the second layer. After drying for 4 hours, draw an appropriate amount of base liquid onto the second layer of the mold, ultrasonicate for 10 minutes, and then place it in a 45°C drying oven for 12 hours. Carefully demold the mold to obtain the soluble microneedles of the four-yellow composition.

[0080] Example 7:

[0081] A soluble microneedle of a four-yellow composition:

[0082] 1. Weigh 1.5 g PVP, 0.2 g CMC-Na, and 8.3 g of the 50 mg / mL DES four-yellow extract obtained by dilution in Example 5 into a beaker, stir for 1 hour, and centrifuge at 6000 rpm for 10 minutes to obtain a mother liquor;

[0083] 2. Weigh 0.5 g of HA into another beaker, stir for 1 hour, and centrifuge at 6000 rpm for 10 minutes to prepare the base solution;

[0084] 3. Use a pipette to draw an appropriate amount of mother liquor onto the microneedle mold, ultrasonicate for 10 minutes, and then place it in a vacuum drying oven (vacuum degree: -0.5Mpa) for 10 minutes. After the vacuum is over, ultrasonicate to remove bubbles, and place it in a 45°C drying oven for 2 hours to obtain the first layer. After drying for 2 hours, draw an appropriate amount of mother liquor onto the first layer of the mold again, ultrasonicate for 10 minutes, and then place it in a 45°C drying oven for 4 hours to obtain the second layer. After drying for 4 hours, draw an appropriate amount of base liquid onto the second layer of the mold, ultrasonicate for 10 minutes, and then place it in a 45°C drying oven for 12 hours. Carefully demold the mold to obtain the soluble microneedles of the four-yellow composition.

[0085] Example 8:

[0086] A soluble microneedle of a four-yellow composition:

[0087] 1. Weigh 1.5 g PVP, 0.2 g CMC-Na, and 8.3 g of the 20 mg / mL DES four-yellow extract obtained by dilution in Example 5 into a beaker, stir for 1 h, and centrifuge at 6000 rpm for 10 min to obtain a mother liquor.

[0088] 2. Weigh 0.5 g of HA into another beaker, stir for 1 hour, and centrifuge at 6000 rpm for 10 minutes to prepare the base solution;

[0089] 3. Use a pipette to draw an appropriate amount of mother liquor onto the microneedle mold, ultrasonicate for 10 minutes, and then place it in a vacuum drying oven (vacuum degree: -0.5Mpa) for 10 minutes. After the vacuum is over, ultrasonicate to remove bubbles, and place it in a 45°C drying oven for 2 hours to obtain the first layer. After drying for 2 hours, draw an appropriate amount of mother liquor onto the first layer of the mold again, ultrasonicate for 10 minutes, and then place it in a 45°C drying oven for 4 hours to obtain the second layer. After drying for 4 hours, draw an appropriate amount of base liquid onto the second layer of the mold, ultrasonicate for 10 minutes, and then place it in a 45°C drying oven for 12 hours. Carefully demold the mold to obtain the soluble microneedles of the four-yellow composition.

[0090] Comparative Example 1

[0091] A fluorescent soluble microneedle:

[0092] 1. Weigh 1.5g PVP, 0.2g CMC-Na, and 8.3g Rhodamine B aqueous solution (fluorescent solution, for observation) into a beaker. Stir for 1 hour, then centrifuge at 6000 rpm for 10 minutes to obtain the mother solution.

[0093] 2. Weigh 0.5 g of HA into another beaker, stir for 1 hour, and centrifuge at 6000 rpm for 10 minutes to prepare the base solution;

[0094] 3. Use a pipette to draw an appropriate amount of mother liquid onto the microneedle mold, ultrasonicate for 10 minutes, and then place it in a vacuum drying oven (vacuum degree: -0.5Mpa) for 10 minutes. After the vacuum is over, ultrasonicate to remove bubbles, and place it in a 45°C drying oven for 2 hours to form the first layer. After drying for 2 hours, draw an appropriate amount of mother liquid onto the first layer of the mold again, ultrasonicate for 10 minutes, and place it in a 45°C drying oven for 4 hours to form the second layer. After drying for 4 hours, draw an appropriate amount of base liquid onto the second layer of the mold, ultrasonicate for 10 minutes, and place it in a 45°C drying oven for 12 hours. Carefully demold the mold to obtain fluorescent soluble microneedles.

[0095] Comparative Example 2

[0096] A blank soluble microneedle:

[0097] 1. Weigh 1.5g PVP, 0.2g CMC-Na, and 8.3g ultrapure water (blank control, used to verify that the substrate has no effect) into a beaker. Stir for 1 hour, then centrifuge at 6000 rpm for 10 minutes to obtain the mother solution.

[0098] 2. Weigh 0.5 g of HA into another beaker, stir for 1 hour, and centrifuge at 6000 rpm for 10 minutes to prepare the base solution;

[0099] 3. Use a pipette to draw an appropriate amount of mother liquid onto the microneedle mold, ultrasonicate for 10 minutes, and then place it in a vacuum drying oven (vacuum degree: -0.5Mpa) for 10 minutes. After the vacuum is over, ultrasonicate to remove bubbles and place it in a 45°C drying oven for 2 hours to form the first layer. After drying for 2 hours, draw an appropriate amount of mother liquid onto the first layer of the mold again, ultrasonicate for 10 minutes, and place it in a 45°C drying oven for 4 hours to form the second layer. After drying for 4 hours, draw an appropriate amount of base liquid onto the second layer of the mold, ultrasonicate for 10 minutes, and place it in a 45°C drying oven for 12 hours. Carefully demold the mold to obtain blank soluble microneedles.

[0100] Test Example 1:

[0101] Testing blank soluble microneedle insertion studies

[0102] Comparative Example 2 was inserted into the ex vivo pig skin, which was fixed in 4% polymethanol for 24 hours, immersed in OCT compound as a freezing medium at room temperature for 2 hours, and sliced. Hematoxylin-eosin (HE) staining was performed, and the insertion condition was observed under a laser scanning fluorescence microscope.

[0103] Results (such as Figure 1 ), it can be observed on the ex vivo pig skin that the blank soluble microneedles of Comparative Example 2 form micron "channels" on the skin, indicating that the microneedles of this formula can enter the dermis layer of the skin (dermis thickness: 10-30μm), so that they can effectively release the active ingredients into the skin and play a certain role.

[0104] Test Example 2:

[0105] Detection of the solubility of fluorescent soluble microneedles:

[0106] The fluorescent soluble microneedle prepared in Comparative Example 1 was placed on the surface of pig skin and pressed with a thumb for 0-5 minutes. The dissolution of the needle tip was photographed using a mechanical microscope at 0 seconds, 10 seconds, 30 seconds, and 5 minutes.

[0107] For the basal layer dissolution experiment, a hydrogel film was added as an external dressing to aid dissolution and adhesion. This film was placed over the fluorescent soluble microneedles after pressing for 5 minutes. Dissolution of the basal layer was observed visually within 0-30 minutes. (Weigh 6% PVA into a beaker, stir at 80°C for 1 hour, centrifuge at 6000 rpm for 10 minutes, remove bubbles with ultrasound, pour into a Petri dish, freeze at -24°C for 6 hours, then remove and thaw at room temperature for 6 hours. Repeat freeze-thaw three times to obtain the hydrogel film.)

[0108] Results (such as Figure 2), when the fluorescent soluble microneedles were not inserted into the pig skin at 0s, the morphology of the fluorescent soluble microneedles was good. The fluorescent soluble microneedles were pressed on the surface of the pig skin for 10s, and the needle tip was observed to have dissolved from the mechanical microscope. When the cumulative insertion time reached 30s, it was obvious that the microneedles had become shorter. During the gradual dissolution process, it was found that the microneedles were swelling. After 5min, the microneedles were completely dissolved. In addition, the basal layer had dissolved within 30min and was in a gel state. It can be seen that the microneedles prepared by this formula have excellent solubility and can achieve synchronous release of effective substances during the dissolution process. Therefore, it has the advantages of not needing to remove the needle body after use, reducing the reuse rate and avoiding cross infection.

[0109] Test Example 3:

[0110] Detection of the morphology of the soluble microneedles of the Sihuang composition:

[0111] The soluble microneedles prepared in Examples 6-8 were placed under a mechanical microscope and the morphology of the microneedles was photographed using a 64x microscope. Figure 3 ), the soluble microneedles of the 100mg / ml four-yellow composition have bent needle tips and incomplete needle shapes; the soluble microneedles of the 20mg / ml four-yellow composition have complete needle shapes but are too brittle and easily broken; while the soluble microneedles of the 50mg / ml four-yellow composition have complete needle shapes and excellent morphology.

[0112] Test Example 4:

[0113] Testing the mechanical properties of the soluble microneedles of the Sihuang composite:

[0114] The mechanical strength of the blank microneedles of Comparative Example 2, and the soluble microneedles of the four-yellow composition of Example 6, Example 7, and Example 8 was determined using a displacement force tester. The displacement force gauge consists of a digital push-pull gauge and an upper swing test bench. The rocker of the upper swing test bench can be rotated to move the mobile tip-shaped force measuring probe vertically downward. The microneedle is placed on a flat rigid base with double-sided tape, with the needle tip facing the probe. When the probe touches the highest point of the microneedle tip, the displacement is recorded as 0 mm. The speed of the probe toward the microneedle is 0.02 mm / s. Record the force and displacement until the probe reaches the preset distance of 0.62 mm.

[0115] Because the skin has a certain elasticity, the microneedle will produce a displacement caused by elastic deformation when inserted into the skin. There will be a turning point at the moment of insertion and then it will tend to be stable. This turning point is the force required for the microneedle to penetrate the skin. Usually, the force required to penetrate the stratum corneum of the skin is 0.1N-0.3N. Figure 4 ), the mechanical properties of the blank microneedle in comparative example 2 are 0.221 N, and the mechanical properties of embodiments 6, 7 and 8 are 0.203 N, 0.216 N and 0.217 N, respectively. It can be seen that both the comparative example and the embodiments meet the conditions for the required force for insertion into the skin.

[0116] Test Example 5:

[0117] Detection of drug loading capacity of soluble microneedles of Sihuang composition:

[0118] The DES four yellow extract prepared according to the steps of Example 5 was diluted to a working concentration of 0.05 mg / mL-0.3 mg / mL, and a full wavelength scan was performed at a wavelength of 200-800 nm.

[0119] Results (such as Figure 5 ) showed that the maximum absorption wavelength of the DES four yellow extract prepared in Example 5 was 274 nm. According to linear fitting, the linear equation y = 2.276x + 0.0475 (y: absorbance; x: concentration mg / mL) was obtained, R 2 =0.9990.

[0120] To determine the DES Sihuang extract content in the microneedles prepared from the Sihuang compositions of Examples 6, 7, and 8, the same batch of microneedles was dissolved in 1 mL of PBS (pH 7.4). After stirring for 1 hour, a homogeneous solution was obtained. The absorbance was measured at 274 nm, and the DES Sihuang extract content in the soluble microneedles of the Sihuang compositions of each example was calculated using a linear equation.

[0121] Results (such as Figure 6 ) showed that the contents of DES Sihuang extract in the Sihuang composition microneedles prepared in Examples 6, 7 and 8 were 25.01 mg, 11.78 mg and 5.54 mg, respectively.

[0122] The experimental results of the above morphology, mechanical properties and drug loading can show that the Sihuang extract after DES extraction is relatively viscous. Therefore, in the process of preparing microneedles, the microneedles of the Sihuang composition prepared in Example 6 have bent needle tips, incomplete needle shapes and decreased mechanical strength. Although the mechanical strength of the Sihuang composition microneedles prepared in Example 8 is good, the drug loading is not high. In summary, the Sihuang composition soluble microneedles prepared in Example 7 have complete needle tips, excellent morphology, and a drug loading of 11.78 mg. The mechanical strength is similar to that of the blank soluble microneedles of Comparative Example 4, which meets the force required for insertion into the stratum corneum of the skin.

[0123] Test Example 6:

[0124] Application of Sihuang composite soluble microneedles:

[0125] 1. Transdermal absorption

[0126] In vitro permeation study, the soluble microneedles of the Sihuang composition prepared in Example 7 were applied to the surface of pig skin, and PBS (pH = 7.4) was used as the culture medium for the receptor chamber. In a Franz diffusion cell system, the skin (3 × 3 cm 2 ) was placed between the donor chamber and a 6-mL receiving cell (preheated to 35°C, rotating at 350 rpm) and protected from light. At pre-set intervals of 0.5, 1, 2, 4, 6, 8, and 12 hours, 3-mL aliquots of sample were removed from the receiving cell and immediately refilled with 3 mL of PBS solution. Finally, the resulting samples were analyzed for the content of the DES Sihuang extract at 274 nm, and the cumulative release was calculated using a linear equation.

[0127] Results (such as Figure 7 ), the soluble microneedles of the Sihuang composition prepared in Example 7 released 18% of the active substance in the first 0.5 hours, while the DES Sihuang extract (prepared in Example 5) released 7%. This indicates that the microneedle tips penetrated the skin, creating a channel for the simultaneous release of active substances. In the early stages of release, the microneedles released rapidly, and throughout the release process, the release from the microneedles remained significantly higher than that from the solution. The maximum release from the Sihuang composition microneedles over 12 hours was 83%, while the maximum release from the DES Sihuang extract over 12 hours was only 59%.

[0128] 2. Skin retention

[0129] After the transdermal absorption test, the excised skin was removed intact, rinsed with warm saline, minced, and then ultrasonically extracted with 4 mL of methanol for 10 minutes three times. The combined extracts were filtered through a 0.45 μm microporous membrane. The skin retention was measured using a UV spectrophotometer at 274 nm.

[0130] Results (such as Figure 8 、 9 ) showed that the skin retention of DES Sihuang extract was 29%, while that of the soluble microneedles of the Sihuang combination was 11%. While the active substances in the solution are mostly retained on the skin's surface, it is difficult for them to penetrate the dermis. However, the channels created by the microneedles allow the active substances to penetrate deep into the skin, truly achieving a therapeutic effect on the affected area.

[0131] 3. Determination of antibacterial activity by soluble microneedle liquid culture method

[0132] The soluble microneedles of the four-yellow composition prepared in Example 7 were sterilized under ultraviolet light for 2 hours. The soluble microneedles of the four-yellow composition were dissolved in 4.9 mL of brain heart infusion broth culture medium to prepare a mother liquor concentration of 6.25 mg / mL. The concentrations of 3.125 mg / mL, 1.563 mg / mL, 0.781 mg / mL, 0.391 mg / mL, and 0.195 mg / mL were set according to the half-concentration dilution method. 100 μL of bacterial solution (1.5×10 5 cells / mL). Place the cells in an anaerobic bag and culture them in a constant temperature incubator for 12 h before smearing and observing.

[0133] Results (such as Figure 10 As shown in the data, at a high concentration of 6.25 mg / mL, virtually no bacteria were observed in the soluble microneedles of the Sihuang composition. At 3.125 mg / mL, corresponding to the MIC concentration of the DES Sihuang extract, very few bacteria were observed. However, at lower concentrations, the bacterial concentration gradually increased. As the working concentration increased, the activity of P. acnes decreased, indicating that the soluble microneedles of the Sihuang composition had a significant antibacterial effect, demonstrating a certain degree of acne-removing efficacy.

[0134] 4. Electron microscopic observation of bacterial morphology using the soluble microneedle of the Sihuang composite

[0135] The soluble microneedles of the four-yellow composition prepared in Example 7 were sterilized under ultraviolet light for 2 hours. The soluble microneedles of the four-yellow composition were dissolved in 4.9 mL of brain heart infusion broth culture medium to prepare a mother liquor concentration of 6.25 mg / mL. The concentrations of 3.125 mg / mL, 1.563 mg / mL, 0.781 mg / mL, 0.391 mg / mL, and 0.195 mg / mL were set according to the half-concentration dilution method. 100 μL of bacterial solution (1.5×10 5 cells / mL). After being placed in an anaerobic bag and cultured in a constant temperature incubator for 6 hours, the above 1mL bacterial solution was taken, 1mL PBS was added, centrifuged at 8000rpm for 5min, the supernatant was discarded, and repeated twice. 2.5% glutaraldehyde was added and mixed evenly, and stored at 4°C overnight. After washing once with PBS, 30wt%, 50wt%, 70wt%, 80wt%, 90wt%, and 100wt% ethanol solutions were added in sequence, centrifuged at 8000rpm for 5min, and gradient dehydration was performed. The bacterial morphology was observed using a scanning electron microscope, and the sample was fixed on an aluminum sheet using double-sided tape, sputter-coated with gold, and then scanned at 15kV. The blank group was not treated and bacteria were cultured with culture medium, and the blank soluble microneedles (Comparative Example 2) were used as the control group.

[0136] Results (such as Figure 11) As shown in the blank group, P. acnes had longer bodies and were more numerous, while the blank microneedle group showed no difference from the blank group, indicating that PVP, CMC-Na, and HA had no inhibitory effect on P. acnes. At the MIC concentration, the four-yellow soluble microneedles prepared in Example 7 produced shorter, curved P. acnes bodies, and were fewer in number, indicating that the DES four-yellow extract has a significant inhibitory effect on P. acnes.

[0137] The four-yellow composition of the present invention has excellent morphology and complete needle shape, dissolution ability, mechanical properties that meet the requirements of insertion into the skin, and a high content of DES four-yellow extract.

[0138] In addition, the soluble microneedles of the four-yellow composition of the present invention have a high transdermal release rate and have a significant inhibitory effect on Propionibacterium acnes, which shows that the DES four-yellow extract can enter the skin through the "channel" created by the microneedles and have a certain acne-removing effect.

[0139] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A soluble microneedle of a four-yellow composition, characterized in that: Prepared using the following method: 1) Mixing aqueous solutions of polyvinyl pyrrolidone, sodium carboxymethyl cellulose and DES four yellow extract, centrifuging to obtain a mother liquor; 2) preparing a 3-7 wt% aqueous solution of hyaluronic acid DES four yellow extract as the base solution; 3) Pour the mother liquid onto the microneedle mold, sonicate, vacuumize, and dry to obtain the first layer; aspirate the mother liquid onto the first layer, sonicate, and dry to obtain the second layer; pour the base liquid onto the second layer, sonicate, dry, and demould to obtain soluble microneedles of the four-yellow composition; In step 1), based on weight, 10-20 parts of polyvinyl pyrrolidone, 1-3 parts of sodium carboxymethyl cellulose, and 75-90 parts of DES four-yellow extract are used. The preparation method of the DES four-yellow extract comprises the following steps: S1: Prepare a deep eutectic solvent: use amino acid as HBA and alcohol or carboxylic acid as HBD, heat and stir to obtain DES; When preparing the DES Scutellaria baicalensis extract, lysine was used as HBA, citric acid was used as HBD, and the molar ratio of lysine:citric acid was 1:4; When preparing the DES Phellodendron amurense extract, lysine was used as HBA, xylitol was used as HBD, and the molar ratio of lysine:xylitol was 1:2; When preparing DES Coptis chinensis extract, arginine was used as HBA, malic acid was used as HBD, and the molar ratio of arginine:malic acid was 1:4; When preparing the DES rhubarb extract, arginine was used as HBA, 1,3-propanediol was used as HBD, and the molar ratio of arginine:1,3-propanediol was 1:2; S2: preparing DES aqueous solution: mixing the DES prepared in S1 with water to obtain a DES aqueous solution; S3: Extraction: Phellodendron amurense, Coptis chinensis, Rhubarb and Scutellaria baicalensis are crushed and sieved, and the DES aqueous solution prepared in S2 is added respectively. The mixture is ultrasonicated at 55-65°C for 30-60 minutes, cooled to room temperature, centrifuged, and the supernatant is dried to obtain the DES four-yellow extract; the weight ratio of DES Phellodendron amurense extract, DES Coptis chinensis extract, DES Rhubarb extract and DES Scutellaria baicalensis extract in the DES four-yellow extract is 1:1:1:

2.

2. The soluble microneedle of the four yellow composition according to claim 1, characterized in that: In S1, the amino acid is selected from at least one of lysine and arginine; the alcohol is selected from at least one of xylitol and 1,3-propylene glycol; and the molar ratio of the HBA to the HBD is 1:2-4.

3. The soluble microneedle of the four yellow composition according to claim 1, characterized in that: In S3, the temperature of the ultrasound is 55-60°C, and the time is 30-40 min; the speed of the centrifugation is 5500-6500 r / min, and the time is 15-25 min; in S3, the specific parameters of the drying are: temperature 55-65°C, vacuum degree -0.3~-1 MPa, and time 10-14 h.

4. The soluble microneedle of the four yellow composition according to claim 1, characterized in that: In step 1), the concentration of the aqueous solution of the DES four yellow extract is 20-100 mg / mL.

5. The soluble microneedle of the four yellow composition according to claim 1, characterized in that: In step 1), the mixing is stirring for 0.5-1.5 hours; in step 1), the centrifugal speed is 5500-6500 r / min for 5-15 minutes.

6. The four-yellow composition soluble microneedle according to claim 1, characterized in that: In step 2), the specific operation of the preparation is: mixing HA and the aqueous solution of DES four yellow extract for 0.5-1.5 hours, centrifuging, and removing bubbles to obtain the base solution.

7. The four-yellow composition soluble microneedle according to claim 1, characterized in that: In step 3), the ultrasonic treatment time is 5-15 minutes; the drying temperature is 40-50° C. and the drying time is 1.5-2.5 hours; the vacuum degree is -0.3 to -1 MPa and the drying time is 5-15 minutes.

8. Use of the soluble microneedles of the four-yellow composition according to any one of claims 1 to 7 in the preparation of anti-acne products.

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