Four-yellow composition soluble microneedle and application thereof

Abstract

The invention provides a four-yellow composition soluble microneedle and application thereof, and relates to the technical field of transdermal drug delivery. The invention relates to a four-yellow composition soluble microneedle, which is prepared by the following preparation method: mixing polyvinylpyrrolidone, sodium carboxymethyl cellulose and an aqueous solution of DES four-yellow extract, and centrifuging to obtain mother liquor; preparing a 3-7wt% hyaluronic acid DES four-yellow extract aqueous solution as a substrate solution; putting the mother liquor into a microneedle mold, performing ultrasonic treatment, then performing vacuum treatment, and drying to obtain a first layer; absorbing mother liquor on the first layer for the second time, and performing ultrasonic treatment and drying to obtain a second layer; and taking the substrate liquid on the second layer, and carrying out ultrasonic treatment, drying and demolding to obtain the four-yellow composition soluble microneedle. The four-yellow composition soluble microneedle prepared by the preparation method disclosed by the invention can break through'obstruction 'of a skin cuticle, so that active substances reach a skin corium layer, and the effects of inhibiting acnes, removing acnes and soothing skin of DES four-yellow extract raw materials are efficiently improved.

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 an application thereof. Background Art

[0002] Flavonoid Chinese medicines such as Scutellaria baicalensis, Phellodendron chinense, Coptis chinensis, and Rhubarb all have the effects of clearing away heat and detoxification, anti-inflammatory and antibacterial, and antioxidant, as well as inhibiting acne, removing acne, repairing and soothing the skin. According to the "Golden Chamber" by Zhang Zhongjing in the Han Dynasty, the Sanhuang Xiexin Decoction, which consists of three herbs, Coptis chinensis, Scutellaria baicalensis, and Rhubarb, is a classic prescription for purging fire and detoxification, which can treat symptoms such as internal evil fire and internal accumulation of blood stasis and heat. In addition, the Huanglian Jiedu Decoction from the "Emergency Prescriptions for the Elbow" is "3 liang of Coptis chinensis, 2 liang of Phellodendron chinense and Scutellaria baicalensis, 14 Gardenia jasminoides, 6 liters of water, decocted to 2 liters, and divided into two doses". It has the function of purging fire and detoxification, and is mainly used to treat all kinds of real heat and fire toxins. It has a significant effect in the surgical treatment of carbuncle and furuncle. According to the ancient prescription, the Sihuang extract, which is made by mixing Phellodendron chinense, Coptis chinensis, Rhubarb, and Scutellaria baicalensis in a certain ratio, can not only improve the effect of acne removal to a certain extent, but also form a unique and innovative combination of Chinese herbal medicine and cosmetics.

[0003] The skin is mainly composed of three layers - the outermost stratum corneum, the middle epidermis and the thickest dermis. The skin is the main barrier to drug delivery via topical routes, and the stratum corneum is like a "wall" in the barrier, which only allows certain molecules, such as lipophilic and low molecular weight drugs to pass through. The relatively low permeability of this layer brings many problems to the design of topical formulations. The most commonly used transdermal drug delivery methods are subcutaneous injection needles and topical ointments. This type of method has the problems of pain caused by injection needles, patients are not very accepting of needles, and topical creams are difficult to penetrate the stratum corneum of the skin, and have low bioavailability.

[0004] In recent years, researchers have studied various topical or transdermal drug delivery systems to improve the penetration of drugs through the skin, such as nanocarrier-loaded topical creams, transdermal patches, and microneedles. Among them, microneedles refer to needle-like structures with a diameter of less than tens of microns and a length of 25-2000μm. Microneedle transdermal drug delivery refers to the use of a certain number of microneedle arrays to pierce the stratum corneum on the skin to produce micron-scale channels that allow drug molecules or even drug particles to be directly introduced into the subcutaneous tissue, thereby achieving drug delivery and effectively improving the utilization rate of active substances. Microneedles are divided into solid microneedles, hollow microneedles, coated microneedles, soluble microneedles, and hydrogel microneedles.

[0005] Dissolving microneedles are made of biodegradable polymer materials (such as hyaluronic acid, polyvinyl pyrrolidone, etc.) and drugs. After piercing the skin, the needle body made of degradable materials will gradually degrade in the microenvironment, and the drug will be released synchronously. The drug molecules pass through the stratum corneum barrier and are absorbed into the human body through the subcutaneous tissue. Dissolving microneedle drug delivery does not require the removal of the needle body after the drug is delivered, which greatly improves patient compliance. At the same time, not reusing microneedles also reduces the risk of cross infection.

[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] In view of the above problems, the present invention provides an optimal formula and preparation method of soluble microneedles of a four-yellow composition using polyvinyl pyrrolidone (PVP), sodium carboxymethyl cellulose (CMC-Na), hyaluronic acid (HA), and DES four-yellow extract as raw materials. In addition, the soluble microneedles of the four-yellow composition can break through the "obstruction" of the stratum corneum of the skin, allowing the active substances to reach the dermis of the skin, effectively improving the efficacy of four-yellow in inhibiting acne, removing acne, and soothing the skin.

[0008] To achieve the above purpose, 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 aqueous solution of DES four yellow extract, centrifuging to obtain mother solution;

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

[0012] 3) taking the mother liquid to the microneedle mold, vacuuming and drying after ultrasonication to obtain the first layer; aspirating the mother liquid on the first layer for the second time, ultrasonicating and drying to obtain the second layer; taking the base liquid on the second layer, ultrasonicating, drying and demoulding to obtain the soluble microneedles of the four yellow composition.

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

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

[0015] More preferably, in step 1), by 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 method for preparing the DES four yellow extract comprises the following steps:

[0020] S1: prepare a low 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, crush and sieve, add the DES aqueous solution prepared in S2, ultrasonicate at 55-65°C for 30-60min, cool to room temperature, centrifuge, take the supernatant and dry it to obtain the 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 being transferred 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 min.

[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 h. 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 h.

[0037] Preferably, in step 2), the specific operation of the configuration is: HA and the aqueous solution of DES four yellow extract are mixed for 0.5-1.5 hours, centrifuged, and bubbles are removed 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), all the ultrasonic times are 5-15 min. More preferably, in step 3), all the ultrasonic times are 10 min.

[0041] Preferably, in step 3), all the drying temperatures are 40-50°C and the drying time is 1.5-2.5 h; further preferably, in step 3), all the drying temperatures are 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 above-mentioned four-yellow composition soluble microneedles in the preparation of acne-removing products.

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

[0045] The preparation process of the soluble microneedle of the four-yellow composition of the present invention is simple, and the dissolution ability is fast and the transdermal ability is good. In addition, the soluble microneedle of the four-yellow composition obtained by loading the DES four-yellow extract into the soluble microneedle can not only solve the problem that the effective substances of the traditional smear ointment only stay on the surface of the skin and are difficult to enter the deep layer of the skin, but also the soluble microneedle studied allows the active substances to reach the dermis of the skin, effectively improving the efficacy of DES four-yellow in inhibiting acne, removing acne and soothing the skin, making it 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 microneedle prepared in Comparative Example 1.

[0048] Figure 3Microscope images of the soluble microneedles of the four yellow compositions prepared in Examples 6-8.

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

[0050] Figure 5 This is a full wavelength scan 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] Fig. 9 This is a comparison chart of the retention amounts of the soluble microneedles of the four-yellow composition prepared in Example 7 and the DES four-yellow extract prepared in Example 5.

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

[0056] Fig.11 This is an electron micrograph of the soluble microneedles of the Sihuang composition prepared in Example 7. (Electron microscopy conditions: magnification: X10000, acceleration voltage: 15KV, working distance: 7.8mm, scale: 1μm). 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 explained below in conjunction with specific embodiments, but the following embodiments are only preferred embodiments of the present invention, not all. Based on the embodiments in the implementation mode, other embodiments obtained by those skilled in the art without making creative work all belong to 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 sources are not specifically limited. The technology and scientific terms used in the embodiments have the meanings 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 the technical field.

[0059] Embodiment 1:

[0060] A DES scutellaria baicalensis extract (lysine: citric acid molar ratio is 1:4, water content is 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 appears. 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 is cooled 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 water, which is the DES Scutellaria baicalensis extract.

[0062] Embodiment 2:

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

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

[0065] Embodiment 3:

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

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

[0068] Embodiment 4:

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

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

[0071] Embodiment 5:

[0072] DES Four Yellow Extract from Phellodendron chinense, Coptis chinensis, Rhubarb and Scutellaria baicalensis

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

[0075] Embodiment 6:

[0076] A four-yellow composite soluble microneedle:

[0077] 1. Weigh 1.5 g PVP, 0.2 g CMC-Na, and 8.3 g 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 solution;

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

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

[0080] Embodiment 7:

[0081] A four-yellow composite soluble microneedle:

[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 solution;

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

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

[0085] Embodiment 8:

[0086] A four-yellow composite soluble microneedle:

[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 hour, and centrifuge at 6000 rpm for 10 minutes to obtain a mother liquor.

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

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

[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) in a beaker. Stir for 1h, and centrifuge at 6000rmp for 10min to obtain the mother solution;

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

[0094] 3. Use a pipette to suck an appropriate amount of mother liquid onto the microneedle mold, ultrasonicate for 10 minutes, and then put it into a vacuum drying oven (vacuum degree: -0.5Mpa) for 10 minutes. After the vacuum is over, ultrasonicate to remove bubbles, and put it into a 45℃ drying oven for 2 hours to obtain the first layer. After drying for 2 hours, suck an appropriate amount of mother liquid onto the first layer of the mold again, ultrasonicate for 10 minutes, and then put it into a 45℃ drying oven for 4 hours to obtain the second layer. After drying for 4 hours, suck an appropriate amount of base liquid onto the second layer of the mold, ultrasonicate for 10 minutes, and then put it into a 45℃ 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) in a beaker. Stir for 1 hour, and centrifuge at 6000rmp for 10 minutes to obtain the mother solution;

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

[0099] 3. Use a pipette to suck an appropriate amount of mother liquid onto the microneedle mold, and after ultrasonic treatment for 10 minutes, put it into a vacuum drying oven (vacuum degree: -0.5Mpa) for 10 minutes. After the vacuum is over, remove bubbles by ultrasonic treatment and put it into a 45℃ drying oven for 2 hours to obtain the first layer. After drying for 2 hours, suck an appropriate amount of mother liquid onto the first layer of the mold again, ultrasonic treatment for 10 minutes, and put it into a 45℃ drying oven for 4 hours to obtain the second layer. After drying for 4 hours, suck an appropriate amount of base liquid onto the second layer of the mold, ultrasonic treatment for 10 minutes, and put it into a 45℃ 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, and then stained with hematoxylin-eosin (HE), 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), allowing it to 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 dissolution experiment of the basal layer, a hydrogel film is needed as an external dressing to assist dissolution and adhesion. It is covered on the fluorescent soluble microneedles after pressing for 5 minutes, and the dissolution of the basal layer is observed with the naked eye within 0-30 minutes. (Weigh 6% PVA in a beaker, stir at 80℃ for 1 hour, centrifuge at 6000rmp for 10 minutes, remove bubbles with ultrasound, pour into a culture dish, put in a -24℃ refrigerator for 6 hours, take out and thaw at room temperature for 6 hours, repeat freezing and thawing 3 times to obtain a hydrogel film.)

[0108] Results (such as Figure 2) shows that at 0s, when the fluorescent soluble microneedles were not inserted into the pig skin, the morphology was good. The fluorescent soluble microneedles were pressed on the surface of the pig skin for 10s, and the needle tips were observed to have dissolved from a mechanical microscope. When the cumulative insertion was 30s, it was clearly seen that the microneedles became 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 dissolution ability and can achieve synchronous release of effective substances during the dissolution process. Therefore, it has the advantages of not having 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 four yellow 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 100 mg / ml four-yellow composition had bent needle tips and incomplete needle shapes; the soluble microneedles of the 20 mg / ml four-yellow composition had complete needle shapes but were too brittle and easily broken; while the soluble microneedles of the 50 mg / ml four-yellow composition had complete needle shapes and excellent morphology.

[0112] Test Example 4:

[0113] Testing the mechanical properties of the soluble microneedles of the four yellow composites:

[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 0mm. The speed of the probe toward the microneedle is 0.02mm / s. Record the force and displacement until the probe reaches a preset distance of 0.62mm.

[0115] Since 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 into the skin and then tend to be stable. This turning point is the force of the microneedle to insert into the skin. Usually, the force required to insert into the stratum corneum of the skin is 0.1N-0.3N. Results (such as Figure 4 ), the mechanical properties of the blank microneedle in comparative example 2 is 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 of the required force for insertion into the skin.

[0116] Test Example 5:

[0117] Detection of drug loading 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 274nm. 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] In order to determine the content of DES four yellow extract in the four yellow composition microneedles prepared in Examples 6, 7 and 8, the same batch of microneedles was dissolved in 1 mL PBS (pH = 7.4). After stirring for 1 hour, a uniform solution was obtained, and the absorbance was measured at 274 nm. According to the linear equation, the content of DES four yellow extract in the soluble microneedles of the four yellow composition of each example was obtained.

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

[0122] It can be seen from the above-mentioned experimental results of morphology, mechanical properties and drug loading 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 microneedles of the Sihuang composition prepared in Example 8 have good mechanical strength, the drug loading is not high. In summary, the soluble microneedles of the Sihuang composition 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 soluble microneedles of Sihuang composition:

[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 medium for the receptor chamber. In a Franz diffusion cell system, the skin (3×3 cm 2 ) was placed between the donor cavity and the 6 mL receiving pool (preheated to 35°C, 350 rpm), protected from light. At pre-set intervals of 0.5, 1, 2, 4, 6, 8 and 12 h, 3 mL aliquots of the sample were taken out from the receiving pool and immediately refilled with 3 mL of PBS solution. Finally, the obtained samples were used to determine the content of DES four yellow extract at 274 nm, and the cumulative release was calculated by a linear equation.

[0127] Results (such as Figure 7 ), the soluble microneedles of the four-yellow composition prepared in Example 7 released 18% in the first 0.5h, while the DES four-yellow extract (prepared in Example 5) released 7%, which shows that the needle tip of the microneedle is inserted into the skin, creating a channel to achieve the synchronous release of effective substances. In the early stage of release, the microneedle release speed is fast, and the release amount of the microneedle is significantly higher than that of the solution throughout the release process. The maximum release amount of the four-yellow composition microneedle in 12h is 83%, while the maximum release amount of the DES four-yellow extract in 12h is only 59%.

[0128] 2. Skin retention

[0129] After the transdermal absorption test, the ex vivo skin was completely removed, the skin was rinsed with warm saline, cut into pieces, and 4 mL of methanol was added. Ultrasonic extraction was performed for 10 min each time, repeated 3 times, and the extracts were combined and filtered through a 0.45 um microporous membrane. The skin retention amount was measured using an ultraviolet spectrophotometer at 274 nm.

[0130] Results (such as Figure 8 , 9 ) showed that the skin retention of DES four-yellow extract was 29%, and the soluble microneedle of the four-yellow combination was 11%. The effective substances in the solution are mostly retained on the skin surface and are difficult to enter the dermis of the skin. The channels formed by the microneedles allow the effective substances to enter the skin and truly have 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, and the mother solution concentration was 6.25 mg / mL. The concentrations were set to 3.125 mg / mL, 1.563 mg / mL, 0.781 mg / mL, 0.391 mg / mL, and 0.195 mg / mL according to the half-concentration dilution method, and 100 μL of bacterial solution (1.5×10 5 cells / mL). The cells were placed in an anaerobic bag and cultured in a constant temperature incubator for 12 h before being plated for observation.

[0133] Results (such as Fig.10 ), almost no bacteria can be seen in the soluble microneedles of the four-yellow composite at a high concentration of 6.25 mg / mL, and very few bacteria can be observed at 3.125 mg / mL, which corresponds to the MIC concentration of the DES four-yellow extract, while the concentration of bacteria gradually increases at low concentrations. As the working concentration increases, the activity of P. acnes becomes worse, indicating that the soluble microneedles of the four-yellow composite have a very obvious antibacterial effect and have the effect of removing acne to a certain extent.

[0134] 4. Electron microscopic observation of the morphology of bacteria using the soluble microneedle of the Sihuang combination

[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, and the mother solution concentration was 6.25 mg / mL. The concentrations were set to 3.125 mg / mL, 1.563 mg / mL, 0.781 mg / mL, 0.391 mg / mL, and 0.195 mg / mL according to the half-concentration dilution method, and 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 aspirated, 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 and drying were performed. The bacterial morphology was observed using a scanning electron microscope, and the sample was fixed on an aluminum sheet using double-sided tape, and coated with gold sputtering, and then scanned at 15kV. The blank group was the group that was not treated and cultured with bacteria in culture medium, and the blank soluble microneedle (Comparative Example 2) was the control group.

[0136] Results (such as Fig.11), the blank group had a longer "body" of P. acnes and a larger number, while the blank microneedle group had no difference from the blank group, indicating that PVP, CMC-Na and HA had no inhibitory effect on P. acnes. The four-yellow composition soluble microneedles prepared in Example 7 had a shorter "body" of P. acnes at the MIC concentration, with a curved shape and a smaller number, indicating that the DES four-yellow extract had 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 amount 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 play 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 preparation method: 1) mixing polyvinyl pyrrolidone, sodium carboxymethyl cellulose and aqueous solution of DES four yellow extract, centrifuging to obtain mother solution; 2) preparing 3-7 wt % aqueous solution of hyaluronic acid DES four yellow extract as base liquid; 3) taking the mother liquid to the microneedle mold, vacuuming and drying after ultrasonication to obtain the first layer; aspirating the mother liquid on the first layer for the second time, ultrasonicating and drying to obtain the second layer; taking the base liquid on the second layer, ultrasonicating, drying and demoulding to obtain the soluble microneedles of the four yellow composition.

2. The four-yellow composition soluble microneedle according to claim 1, characterized in that: The preparation method of the DES four yellow extract described in step 1) comprises the following steps: S1: prepare a low eutectic solvent: use amino acid as HBA and alcohol or carboxylic acid as HBD, heat and stir to obtain DES; S2: preparing DES aqueous solution: mixing the DES prepared in S1 with water to obtain a DES aqueous solution; S3: Extraction: Take Phellodendron amurense, Coptis chinensis, Rhubarb and Scutellaria baicalensis, crush and sieve, add the DES aqueous solution prepared in S2, ultrasonicate at 55-65°C for 30-60min, cool to room temperature, centrifuge, take the supernatant and dry it to obtain the DES four-yellow extract.

3. The four-yellow composition soluble microneedle according to claim 2, 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.

4. The four-yellow composition soluble microneedle according to claim 2, 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 is 55-65°C, vacuum degree is -0.3 to -1 MPa, and time is 10-14 h.

5. The four-yellow composition soluble microneedle according to claim 1, characterized in that: In step 1), according to the weight percentage, polyvinyl pyrrolidone is 10-20 parts, sodium carboxymethyl cellulose is 1-3 parts, and DES four yellow extract is 75-90 parts.

6. The four-yellow composition soluble microneedle 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.

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

8. The four-yellow composition soluble microneedle according to claim 1, characterized in that: In step 2), the specific operation of the configuration is: HA and the aqueous solution of DES four yellow extract are mixed for 0.5-1.5 hours, centrifuged, and bubbles are removed to obtain the base solution.

9. The four-yellow composition soluble microneedle according to claim 1, characterized in that: In step 3), all the ultrasonic treatment times are 5-15 min; all the drying temperatures are 40-50° C. and the drying time is 1.5-2.5 h; the vacuum degree of the vacuum is -0.3 to -1 MPa and the drying time is 5-15 min.

10. Use of the soluble microneedles of the four yellow composition according to any one of claims 1 to 9 in the preparation of anti-acne products.

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