Cinnamomum cassia volatile oil-loaded microneedle acupoint patch and application thereof in treatment of osteoarthritis

Abstract

This invention discloses a microneedle acupoint patch loaded with cinnamon volatile oil and its application in the treatment of osteoarthritis. The microneedle acupoint patch comprises a needle tip layer and a backing layer. The needle tip layer uses polyvinylpyrrolidone and chondroitin sulfate as a matrix to encapsulate a mixed oil phase prepared from cinnamon volatile oil and 1-butyl-3-methylimidazolium hexafluorophosphate. 1-Butyl-3-methylimidazolium hexafluorophosphate improves the compatibility of cinnamon volatile oil with the matrix, further increasing the content of cinnamon volatile oil in the microneedles. The microneedle acupoint patch was used to treat osteoarthritis in mice. The results showed that the microneedle acupoint patch significantly reduced swelling at the knee joint of mice, improved pain threshold and pain scores, and significantly reduced cartilage wear and inflammation, demonstrating high safety. Therefore, this invention provides a soluble microneedle patch loaded with cinnamon volatile oil with a simpler preparation method and higher drug loading capacity, which has important reference value for the clinical treatment of osteoarthritis.

Description

Technical Field

[0001] This invention belongs to the field of osteoarthritis treatment technology, specifically, it relates to a microneedle acupoint patch loaded with cinnamon volatile oil and its application in the treatment of osteoarthritis. Background Technology

[0002] Osteoarthritis (OA) is a chronic, progressive, and destructive inflammatory disease of articular cartilage, synovium, and their appendages. Its main clinical manifestations include joint swelling, chronic pain, and limited mobility, causing significant disruption to patients' work and daily lives, imposing a substantial social burden, and resulting in a high disability rate. The article "Research Progress on the Anti-inflammatory and Anti-tumor Effects and Mechanisms of Cinnamon Oil" points out that the volatile oil extracted from the dried branches and leaves of cinnamon through steam distillation has anti-arthritis effects, but further research into enhancing the deep processing of cinnamon volatile oil to improve its efficacy in treating arthritis holds broad research potential.

[0003] Microneedle transdermal drug delivery is a novel drug delivery technology. Its basic principle is to use microneedles to penetrate the stratum corneum of the skin, creating tiny channels on the skin surface. This allows drugs to directly enter the epidermis or dermis, and even reach the systemic circulation, achieving effective drug delivery. Traditional transdermal drug delivery methods have low drug penetration efficiency through the stratum corneum, while microneedle transdermal drug delivery can increase transdermal throughput by several orders of magnitude, improving drug bioavailability. Furthermore, the short length of the microneedles generally avoids nerve endings when penetrating the skin, so users typically experience only mild tingling or almost no sensation. This offers significant advantages over injection-based drug delivery and has become a hot research topic in recent years. However, current microneedle formulations suffer from problems such as low drug loading capacity and poor biocompatibility of matrix materials. Existing acupoint stimulation methods also have drawbacks, including difficulties in drug delivery, significant individual variability in stimulation effects, and challenges in controlling precision and dosage. Chinese patent application CN114146048A discloses a hydrogel microneedle integrating acupuncture and medication. The microneedle matrix is ​​a hydrogel formed by the cross-linking of gelatin and tannic acid, and the matrix encapsulates a drug, namely cinnamon oil nanocapsules, which can be used for clinical treatment of acupoint therapy. However, the drug loading capacity of the microneedle is limited by the proportion of cinnamon oil nanocapsules added to the matrix and the proportion of cinnamon oil added to the nanocapsules, resulting in a low drug loading capacity. At the same time, if the capsule wall material is too stable or too tightly bound to the matrix, the drug may not be completely released within the expected time. Therefore, this invention provides a soluble microneedle carrying cinnamon volatile oil with a simpler preparation method and a higher drug loading capacity, which is of great significance. Summary of the Invention

[0004] The purpose of this invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide a microneedle acupoint patch containing cinnamon volatile oil, which can be used for local administration to treat osteoarthritis.

[0005] The second objective of this invention is to provide the application of the above-mentioned microneedle acupoint patch in the preparation of products for treating osteoarthritis.

[0006] The above-mentioned objective of this invention is achieved through the following technical solution: This invention provides a microneedle acupoint patch, which includes a backing layer and a needle tip layer; the preparation method of the needle tip layer includes the following steps: S1. A matrix material prepared from chondroitin sulfate and polyvinylpyrrolidone; S2. A mixed oil phase was prepared by reacting cinnamon volatile oil with 1-butyl-3-methylimidazolium hexafluorophosphate; S3. Mix the mixed oil phase with emulsifier and water, then mix with the matrix material to obtain a drug-containing matrix material solution, and pour it into a microneedle mold.

[0007] This invention provides a microneedle acupoint patch loaded with cinnamon volatile oil, which can improve osteoarthritis. The patch includes a needle tip layer and a backing layer. The needle tip layer uses polyvinylpyrrolidone and chondroitin sulfate as a matrix to encapsulate the active pharmaceutical ingredient (cinnamon volatile oil). To improve the compatibility between the cinnamon volatile oil and the matrix, this invention investigated the effects of several ionic liquids on their compatibility. The results showed that, compared to the other three ionic liquids, treatment with 1-butyl-3-methylimidazolium hexafluorophosphate significantly improved the compatibility between the cinnamon volatile oil and the matrix, further increasing the content of cinnamon volatile oil in the microneedle layer. The microneedle acupoint patch provided by this invention has a high drug loading capacity, allowing it to penetrate the stratum corneum and reach the dermis, achieving transdermal drug delivery and releasing the active pharmaceutical ingredient for precise drug delivery and acupoint stimulation.

[0008] Furthermore, in step S1, the mass ratio of chondroitin sulfate to polyvinylpyrrolidone is 2:1. Chondroitin sulfate (CS) and polyvinylpyrrolidone (PVP) have good rigidity and are soluble (dissolving almost completely within 3 minutes). The resulting needle tip possesses certain mechanical properties, exhibits optimal puncture performance, and spontaneously dissolves to release the drug upon piercing the stratum corneum of the skin. This material is also biocompatible, producing no adverse reactions after dissolution and absorption.

[0009] Furthermore, in step S2, the concentration of cinnamon volatile oil in the mixed oil phase is 160–170 mg / mL.

[0010] Preferably, the concentration of cinnamon volatile oil in the mixed oil phase in step S2 is 166 mg / mL.

[0011] Furthermore, the emulsifier in step S3 is composed of Tween-80 and anhydrous ethanol. Modifying the active pharmaceutical ingredient with emulsifiers and co-emulsifiers can improve the loading capacity and compatibility of the active pharmaceutical ingredient in the needle tip layer.

[0012] Furthermore, the volume ratio of Tween-80 to anhydrous ethanol is 3:1.

[0013] Furthermore, in step S3, the ratio of the mixed oil phase to the emulsifier and water is 6.5–7.5:0.8–1.2:1.8–2.2.

[0014] Preferably, the ratio of the mixed oil phase to the emulsifier and water in step S3 is 7:1:2.

[0015] Furthermore, in step S3, the proportion of matrix material in the drug-containing matrix material solution is 70-80%.

[0016] Preferably, in step S3, the matrix material in the drug-containing matrix material solution accounts for 75%.

[0017] Furthermore, the backing layer is made of polyvinyl alcohol. Polyvinyl alcohol (PVA) has moderate toughness and rigidity, achieving the effect of bending without breaking. It is also breathable and oxygen-permeable. Wounds caused by it heal within 1 hour in rat skin experiments. It also has a certain degree of biocompatibility and does not produce adverse reactions such as allergies after contact with the skin.

[0018] Preferably, the backing layer is prepared from 25% polyvinyl alcohol.

[0019] Furthermore, the method for preparing the cinnamon volatile oil is to pulverize cinnamon to 20-80 mesh, soak it, extract it with ultrasonic assistance, and distill it at a material-to-liquid ratio of 1:10 for 4-8 hours.

[0020] Furthermore, the microneedle mold is prepared from polydimethylsiloxane.

[0021] Furthermore, the needle tip length of the needle tip layer can reach 400-800 μm or more, and after puncturing the skin, it can penetrate the stratum corneum to reach the dermis.

[0022] Preferably, the needle tip layer has a needle length of 570 μm, a needle tip distance of 700 μm, and a microneedle acupoint patch diameter of 17.5 mm.

[0023] Specifically, the present invention also provides a feasible method for preparing a microneedle acupoint patch, comprising the following steps: S1. Dissolve chondroitin sulfate and polyvinylpyrrolidone thoroughly, melt, swell and stir evenly, and sonicate for 20 minutes to obtain a matrix material solution; S2. Weigh out Tween-80 and anhydrous ethanol in a 3:1 ratio, mix well to prepare a mixed emulsifier; S3. Cinnamon volatile oil was mixed with ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate to form a mixed oil phase with a concentration of 166 mg / mL; S4. Mix the mixed oil phase, water, and mixed emulsifier in a ratio of 7:1:2, take 1.25 mL of the mixture and add it to 3.75 mL of the matrix material solution, mix well to obtain the drug-containing matrix material solution; S5. Prepare a 25% aqueous solution of polyvinyl alcohol backing material; S6. Take the solution from step S4 and pour it evenly onto the polydimethylsiloxane microneedle mold, at 3500 r·min. -1 Centrifuge under certain conditions to ensure uniform filling into the mold, then pour in an aqueous solution of the backing material, dry, and demold to obtain the drug-containing microneedle patch.

[0024] The present invention further applied the microneedle acupoint patch prepared above to treat osteoarthritis in mice. The results showed that the microneedle acupoint patch group could significantly reduce swelling at the knee joint of mice, and the pain threshold and pain score of mice were improved. The degree of cartilage wear and inflammation were also significantly reduced. The safety assessment results also showed that the microneedle acupoint patch of the present invention did not show drug toxicity in the liver and kidneys, and had high safety.

[0025] Therefore, the present invention also provides the application of the above-mentioned microneedle acupoint patch in the preparation of products for treating osteoarthritis.

[0026] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a microneedle acupoint patch loaded with cinnamon volatile oil and its application in the treatment of osteoarthritis. The microneedle acupoint patch includes a needle tip layer and a backing layer. The needle tip layer uses polyvinylpyrrolidone and chondroitin sulfate as a matrix to encapsulate a mixed oil phase prepared from cinnamon volatile oil and 1-butyl-3-methylimidazolium hexafluorophosphate. Treatment with 1-butyl-3-methylimidazolium hexafluorophosphate improves the compatibility of cinnamon volatile oil with the matrix, further increasing the content of cinnamon volatile oil in the microneedles. The microneedle acupoint patch was used to treat osteoarthritis in mice. The results showed that the microneedle acupoint patch significantly reduced swelling at the knee joint of mice, improved pain threshold and pain scores, and significantly reduced cartilage wear and inflammation, with high safety. Therefore, this invention provides a soluble microneedle acupoint patch loaded with cinnamon volatile oil with a simpler preparation method and higher drug loading capacity, which has important reference value for the clinical treatment of osteoarthritis. Attached Figure Description

[0027] Figure 1 A schematic diagram of a microneedle acupoint patch.

[0028] Figure 2 This is a top view diagram of a microneedle acupoint patch.

[0029] Figure 3 The image shows the morphology of the microneedle acupoint patch under an optical microscope.

[0030] Figure 4A scanning electron microscope (SEM) diagram of a microneedle acupoint patch.

[0031] Figure 5 This is a schematic diagram illustrating the in vivo dissolution of the microneedle acupoint patch. Note: From left to right, the dissolution time is 0 min, 1 min, 3 min, and 5 min, respectively.

[0032] Figure 6 A diagram showing the breaking force test of the needle tip of a microneedle acupoint patch (14 needles).

[0033] Figure 7 Statistical chart of the results of microneedle acupuncture with Parafilm.

[0034] Figure 8 XPS energy dispersive spectroscopy scan of microneedle acupoint patches.

[0035] Figure 9 Fourier transform infrared spectroscopy analysis of microneedle acupoint patches.

[0036] Figure 10 This is a standard curve of linearity in high performance liquid chromatography methodology.

[0037] Figure 11 This is a schematic diagram of the specificity experiment in the high performance liquid chromatography methodology.

[0038] Figure 12 A statistical chart comparing the diameter of SD rats treated with microneedle acupoint patches.

[0039] Figure 13 A statistical chart comparing the circumference of SD rats treated with microneedle acupoint patches.

[0040] Figure 14 A statistical chart showing the pain scores of SD rats after microneedle acupoint patch application.

[0041] Figure 15 Pain threshold detection in SD rats after microneedle acupoint patch application.

[0042] Figure 16 HE staining image of liver in SD rats after microneedle acupoint patch application (20×). Note: From left to right in the image, the results are for the normal group, model group, and microneedle group.

[0043] Figure 17 HE staining image of the kidneys of SD rats after microneedle acupoint patch application (10×). Note: From left to right in the image, the results are for the normal group, model group, and microneedle group.

[0044] Figure 18 Comparative CT images of the knee joints of SD rats after microneedle acupoint patch application. Note: From left to right in the images, the results are for the normal group, the model group, and the microneedle group.

[0045] Figure 19 This is a schematic diagram illustrating the skin recovery of SD rats after microneedle acupoint patch application.

[0046] Figure 20 The diagram shows the regulatory effects of microneedle acupoint patches on inflammatory factors IL-1β, IL-6, IL-8, and TNF-α in SD rats. Detailed Implementation

[0047] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0048] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0049] Example 1: Extraction of Cinnamon Volatile Oil Cinnamon was purchased from Zhongrunkang Traditional Chinese Medicine Planting Cooperative in Luoding City. The volatile oil of cinnamon was extracted using steam distillation for subsequent microneedle preparation experiments. The specific method was as follows: cinnamon was crushed to 20-80 mesh using a grinder, soaked in pure water for 30 minutes, and then subjected to ultrasonic-assisted extraction for 40 minutes at a power of 300 W for 6.0 h, with a liquid-to-solid ratio of 1:10 (g / mL). The pale yellow, pungent-smelling oily liquid at the top of the distillation was collected, which is the volatile oil of cinnamon.

[0050] Example 2: Preparation of Microneedle Acupoint Patches 1. Preparation of microneedle acupoint patches (1) Weigh 0.8g chondroitin sulfate (CS) and 0.4g polyvinylpyrrolidone (PVP) (mass ratio of 2:1) into a beaker, add 3.25mL of pure water, dissolve and melt, swell and stir evenly until completely dissolved and swollen, sonicate for 20 minutes to obtain a matrix material solution for later use.

[0051] (2) Weigh Tween-80 and anhydrous ethanol in a volume ratio of 3:1 (Km value) and mix them to obtain a mixed emulsifier (Smix).

[0052] (3) Cinnamon volatile oil was mixed with ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate (BMIMPF6) to form a mixed oil phase with a cinnamon volatile oil concentration of 166 mg / mL.

[0053] (4) Mix the oil phase, water phase (pure water), and mixed emulsifier in a ratio of 7:1:2, take 1.25 mL of the mixture and add it to 3.75 mL of the matrix material solution, mix well to obtain the drug-containing matrix material solution.

[0054] (5) Take polyvinyl alcohol (PVA) and add pure water to prepare a 25% polyvinyl alcohol backing material aqueous solution for later use.

[0055] (6) Take the drug-containing matrix material solution from (4) and pour it evenly onto the PDMS (polydimethylsiloxane) microneedle mold, at 3500 r·min -1 Centrifuge for 5 minutes under the specified conditions, then rotate 180° and centrifuge for another 5 minutes to ensure even distribution into the mold. After centrifugation, scrape off the upper layer of drug-containing matrix material solution, pour in a 25% polyvinyl alcohol (PVA) backing material aqueous solution, and dry in a desiccator at room temperature for 24 hours. Demold to obtain the drug-containing microneedle patch.

[0056] 2. Effect of ionic liquids on the volatile oil content of cinnamon in microneedles The 1-butyl-3-methylimidazolium hexafluorophosphate in step (3) above was replaced with 1-dodecyl-3-methylimidazolium tetrafluoroborate, 1-tetradecyl-3-methylimidazolium chloride, and 1-tetradecyl-3-methylimidazolium hexafluorophosphate, respectively. The results are shown in Table 1. It is shown that 1-butyl-3-methylimidazolium hexafluorophosphate is more suitable for emulsification with cinnamaldehyde in room temperature ionic liquids of imidazolium, and the emulsion formed has the highest content of cinnamaldehyde.

[0057] Table 1. Cinnamaldehyde absorption area of ​​emulsions formed by various ionic liquid types

[0058] Example 3 Characterization and evaluation of microneedle acupoint patches 1. Appearance evaluation of microneedle patches ① The morphology and dimensions of the microneedle acupoint patch prepared in Example 2 were observed and measured using a stereomicroscope. The microneedle acupoint patch was placed on the sample stage, and its overall morphology was observed using a stereomicroscope. Figure 1-3 As shown.

[0059] ② The microstructure was observed and the microneedle size was measured using a ZEISS scanning electron microscope. The results are as follows: Figure 4 As shown, the needle length is 570 μm, the needle tip distance is 700 μm, and the microneedle patch diameter is 17.5 mm.

[0060] 2. Evaluation of the dissolution performance of microneedle patches The microneedle acupoint patch prepared in Example 2 was subjected to an in vitro skin dissolution experiment. Skin from the back of rats was taken, and the skin fascia was cleaned with anhydrous ethanol. The microneedle patch was then pressed onto the in vitro skin, and removed at 0 min, 1 min, 3 min, and 5 min. The dissolution was observed under a microscope. The results are as follows: Figure 5 As shown, the microneedles dissolved almost completely within 3 minutes.

[0061] 3. Mechanical strength test of microneedle patches ① The fracture force of the needle tip was tested using a texture analyzer, model TA-XT plus, manufactured by Stable Micro Systems. The test conditions were: Test Mode: Compression; Pre-Test Speed: 0.8 mm / sec; Test Speed: 0.1 mm / sec; Post-Test Speed: 10 mm / sec; Target Mode: Distance / 0.8 mm; Trigger Type: Auto Force; Trigger Force: 0.05 N. The test results are as follows: Figure 6 As shown (the example is the total breaking force of 14 needles), the average breaking force of a single needle in the microneedles was measured to be 1.51N.

[0062] ② Parafilm membrane experiment: Parafilm membranes are widely used in human skin models due to their elastic modulus and insertion resistance, which are similar to those of skin. Qualified microneedles can successfully penetrate the first two layers of Parafilm, but differences in insertion depth can be observed in the third and fourth layers when microneedles prepared from different matrix materials are used.

[0063] A. The microneedle patch (CS:PVP=2:1) ​​prepared in Example 2 of this application. B. Replace CS:PVP=2:1 with S-97 (polymethyl vinyl maleic anhydride copolymer, Gantrez S-97):HPMC (hydroxypropyl methylcellulose)=2.5:1, and prepare the microneedle patch using the same method as in Example 2. C. Replace CS:PVP=2:1 with S-97:PVP=1:2, and prepare the microneedle patch using the same method as in Example 2.

[0064] The microneedle patch prepared in Example 2 was pressed with the thumb to pierce into the four overlapping layers of Parafilm membrane. The number of membrane puncture layers and the perforation rate were observed under a microscope, and the perforation rate was statistically analyzed. The results are as follows: Figure 7 As shown, in the Parafilm membrane puncture experiment, the microneedles with CS:PVP=2:1, S-97:HPMC=2.5:1, and S-97:PVP=1:2 all exhibited a perforation rate of 90% in the first two Parafilm membrane layers. In the third layer, the CS:PVP=2:1 and S-97:PVP=1:2 formulations still maintained a perforation rate above 80%, while the S-97:HPMC=2.5:1 formulation decreased to around 70%. In the fourth layer, the perforation rates of all three advantageous formulations were below 50%. Therefore, the CS:PVP=2:1 and S-97:PVP=1:2 formulations yielded better results.

[0065] 4. Microscopic elemental characterization of the microneedle patches was performed using an X-ray photoelectron spectroscopy (XPS) instrument, specifically the Thermo Fisher Scientific K-Alpha. The testing conditions were: a vacuum level of 5 × 10⁻⁶ in the analytical chamber. -10 The excitation source was Alka rays (hv = 1486.68 eV), the operating voltage was 15 kV, the filament current was 10 mA, and signal accumulation was performed for 5-10 cycles. The passing energy was 50 eV, the step size was 0.1 eV, and charge correction was performed using the binding energy C1s = 284.80 eV as the energy standard. The test results are as follows: Figure 8 As shown, the drug-loaded microneedles prepared in Example 2 contain phosphorus and fluorine elements, which are unique elements contained in the ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate, indicating that the ionic liquid microemulsion was successfully combined with the microneedle matrix.

[0066] Fourier transform infrared spectroscopy analysis was performed on the three prescriptions, and the infrared spectra were obtained from the results. Figure 9 As can be seen from the data, the common functional groups include alkyl CH stretching vibration (~2950 cm-1), ether bond (COC) and hydroxyl (OH) vibration (~1070-1100 cm-1, ~3400 cm-1), CH2 / CH3 bending vibration and some aromatic ring skeleton vibration (~1440-1496 cm-1); while among the differences, in formulation a (CS:PVP=2:1) ​​there is halogen bond CF vibration (~846 cm-1), which comes from hexafluorophosphate in ionic liquid, and the hydroxyl group of chondroitin sulfate in formulation (a) enhances the hydroxyl peak (3445 cm-1); in formulations b (S-97:PVP=1:2) and c (S-97:HPMC=2.5:1) there is anhydride C=O vibration (~1770 cm-1), which comes from polymethyl vinyl maleic anhydride copolymer. No obvious halogen bond CF vibration (~846 cm-1) was observed in formulations (b) and (c), which may be because the microneedle matrix material in formulations (b) and (c) binds weakly to the ionic liquid compared to formulation (a).

[0067] Combined with other performance evaluations, the experimental results show that the selected final formulation CS:PVP=2:1 has the best overall performance.

[0068] 5. Detection of cinnamaldehyde content in microneedles ① Take the microneedle patch, gently scrape the needle tip off into a 5mL beaker with a blade, add 3mL of pure water, sonicate to dissolve the needle tip, add 2mL of methanol, and filter the solution through a 0.22μm microporous membrane to obtain the test solution.

[0069] ② Use 0.2% phosphoric acid as mobile phase A and acetonitrile as mobile phase B; the detection wavelength is 254 nm; the flow rate is 1.0 mL / min. -1 The column temperature was 30°C; the injection volume was 20 μL; a C18 reversed-phase column was used; gradient elution was performed according to the elution program in Table 2.

[0070] Table 2 Gradient elution program

[0071] ③Precision test: According to the Chinese Pharmacopoeia (2025) standard, cinnamaldehyde reference standard was prepared into a reference solution with a concentration of 0.626 mg / mL. The solution was injected 6 times continuously under liquid chromatography conditions. The RSD of the concentration measurement was 0.42%, and the relative standard deviation was not greater than 2.0%.

[0072] ④Stability test: Take the test solution from ①, store it at room temperature, and measure it under chromatographic conditions at 0, 2, 4, 8, 12 and 24 h respectively. Record the peak area of ​​the microneedle sample and calculate the RSD of the peak area as 0.82%. The results show that the test solution is stable at room temperature for 24 h.

[0073] ⑤ Linearity Examination: Cinnamon reference standard was prepared into the following concentrations with methanol: 0.631 mg / mL, 0.252 mg / mL, 0.063 mg / mL, and 0.006 mg / mL, respectively. Peak areas were measured, and a standard curve was plotted as y = 49751x + 42440, R0. 2 =0.9996, the result is as follows Figure 10 As shown.

[0074] ⑥ Recovery analysis: Weigh 0.2 g each of CS, PVP, and PVA, and add 20 mL of pure water to each. Prepare the test solutions separately. Accurately weigh 19.75 mg of cinnamaldehyde and dissolve it in 10 mL of methanol. Mix the test solutions with methanol stock solutions at ratios of 1:5, 1:10, and 1:20, and then analyze the recovery rate using HPLC and calculate the RSD. The average RSD was 0.19%.

[0075] ⑦ Specificity Experiment: Weigh 0.2g each of CS, PVP, and PVA, add 20mL of pure water to each, and separately take the cinnamaldehyde reference solution from ③. Perform HPLC analysis to observe whether the peak position affects cinnamaldehyde. Results are as follows: Figure 11 As shown, the retention time of various substances does not affect the detection of cinnamaldehyde, and the retention time of cinnamaldehyde is about 18 minutes.

[0076] Example 4: Efficacy Evaluation of Microneedle Acupoint Patches 1. Establishment of Animal Disease Model: Six-week-old SD rats, with equal numbers of males and females, were divided into three groups: a normal group, a model group, and a microneedling group. They were acclimatized for two weeks. The microneedling group and the model group underwent anterior cruciate ligament transection to establish the model. Before the modeling experiment, the animals' anesthesia and respiratory system tubing was checked, and isoflurane (purchased from Ruiward Life Science Co., Ltd.) was added to the anesthesia machine. Anesthesia was induced using 3% isoflurane, and the rats were placed in the induction box until complete anesthesia. The rats were removed from the induction box and placed supine on a sterile operating table. Anesthesia was maintained using a 1.5% isoflurane mask. The right knee joint of the rat was shaved and prepared. After double disinfection with alcohol and iodine, a longitudinal incision of approximately 2 cm was made with a scalpel. After exposing the knee joint, the patellar ligament was transversely incised with a scalpel, and the anterior and posterior cruciate ligaments and medial collateral ligament were further transcribed. After surgery, the drawer test of the rat's right knee joint was confirmed to be positive (the rat's knee joint was flexed at 90 degrees, and the thigh was held with one hand while the upper part of the lower leg was held with the other hand, and a pulling and pushing motion was performed forward. A positive result was indicated by significant anterior displacement of the tibial tuberosity, indicating that the anterior cruciate ligament had been severed). The patellar ligament and rat skin were sutured layer by layer. Postoperatively, the rats were forced to run for 30 minutes daily using a roller treadmill for two weeks. When joint wear and tear occurred, the knee joint swelled, and the rats showed signs of pain, the model was considered successfully established.

[0077] 2. Animal administration: The microneedle group was given the drug once every two days. The hair of the rat knee joint was shaved clean with a shaver. The microneedle acupoint patch prepared in Example 2 was pressed on the rat knee joint. After the needle tip was completely dissolved, the microneedle patch was removed. The above steps were continued for 8 weeks. The normal group and the model group were not treated. After 8 weeks, the following indicators were tested to evaluate the efficacy.

[0078] 3. Measurement of diameter and circumference at the rat's knee joint ① The circumference of the rat's knee joint was measured using a tape measure. The tape measure was used to encircle the rat's knee joint, and the reading was obtained. The results are as follows: Figure 12 As shown, microneedling significantly reduced swelling at the knee joint of mice, indicating that microneedling intervention improved the degree of swelling at the knee joint of rats.

[0079] ② Measure the diameter of the rat's knee joint at its largest point using vernier calipers; the reading is then obtained. The results are as follows: Figure 13 As shown, microneedle treatment significantly reduced swelling at the knee joint in mice and improved the degree of swelling at the knee joint in rats.

[0080] 4. Pain score of rats ① The rat pain score was assessed using the RGS (Rat Grimace Scale). Four aspects were evaluated: eye socket constriction, nose / cheek bulging, ear position change, and whisker change. The severity of these four aspects was observed, and scores were recorded and tallied. The results are as follows: Figure 14 As shown, compared with the model group, the total RGS pain score of rats was reduced after microneedle intervention.

[0081] ② The pain threshold of rats was tested using a small animal thermal pain meter. The temperature was set to 55℃. The rat was placed on the hot plate and the timing began immediately. The timing was stopped when the rat started licking its heel. The time was recorded. The results are as follows: Figure 15 As shown, compared with the model group, the reaction time of rats on the hot plate increased after microneedling intervention, indicating that microneedling intervention improved the pain threshold of rats to a certain extent and reduced pain compared with the model group.

[0082] 5. Evaluation of hepatorenal toxicity of microbes in rats: After sacrifice, the liver and kidney organs of rats were preserved by immersion in 4% paraformaldehyde and embedded in paraffin. After embedding, tissue sections were prepared for HE staining to observe changes in liver and kidney cell morphology and evaluate hepatorenal toxicity. The results are as follows: Figure 16 , 17 As shown, the microneedle acupoint patch prepared by the present invention has high safety and the morphology of liver and kidney cells did not change.

[0083] 6. Observation of articular surface wear in rats after microneedling: The knee joint of rats was scanned using a MicroCT scanner (model NMC-200, manufactured by Pingsheng Medical Technology). The following parameters were set: radial field of view (FOV): 100 mm; maximum axial scan range: 250 mm; scan speed: fastest 4 s / bed; reconstructed pixel size: Min. 2 μm; spatial resolution: <7.5 μm@10% MTF. The results are as follows: Figure 18 As shown in the MicroCT images, the knee joints of rats in the normal group had regular morphology, smooth and intact articular surfaces, and no obvious bone hyperplasia or deformity. Compared with the normal group, the knee joints of rats in the model group had disordered morphology and structure, with obvious bone hyperplasia at the joint margins and rough and irregular articular surfaces, exhibiting typical osteoarthritis-like bony structural abnormalities. Compared with the model group, the bone structural abnormalities of the rat knee joints were improved to a certain extent after microneedling intervention. The articular surfaces were smoother than those in the model group, the degree of bone hyperplasia and deformity was significantly reduced, and the trochlear groove of the femur was more obvious than that in the model group, indicating that microneedling has a certain ameliorative effect on osteoarthritis.

[0084] 7. Observation of skin recovery in rats after microneedling: Skin recovery in rats was observed at 0 min, 15 min, 30 min, 1 h, and 2 h after the drug administration. Results are as follows: Figure 19 As shown, after five minutes of removal, half of the needle marks disappeared within 15 minutes, and there were no obvious needle marks after 30 minutes. Furthermore, no irritation reactions such as redness, swelling, or erythema occurred after 1 hour and 2 hours, indicating that the microneedle patch has good biocompatibility with the skin.

[0085] 8. The regulatory effect of microneedling on inflammatory factors IL-1β, IL-6, IL-8, and TNF-α in SD rats. Results are as follows... Figure 20 As shown, the microneedles prepared in Example 2 of this invention improved the expression of key proteins in the knee joint of SD rats. In the detection of inflammatory factors IL-1β, IL-6, IL-8, and TNF-α, there were significant differences between the normal group and the model group. P <0.001), while after microneedling intervention, the level of IL-1β in the microneedling group was reduced compared with that in the model group ( P <0.05), and similarly decreased levels were observed in IL-8 and TNF-α. P <0.01).

Claims

1. A microneedle acupoint patch, characterized in that, The microneedle acupoint patch comprises a backing layer and a needle tip layer; the preparation method of the needle tip layer includes the following steps: S1. A matrix material prepared from chondroitin sulfate and polyvinylpyrrolidone; S2. A mixed oil phase was prepared by reacting cinnamon volatile oil with 1-butyl-3-methylimidazolium hexafluorophosphate; S3. Mix the mixed oil phase with emulsifier and water, then mix with the matrix material to obtain a drug-containing matrix material solution, and pour it into a microneedle mold.

2. The microneedle acupoint patch according to claim 1, characterized in that, In step S1, the mass ratio of chondroitin sulfate to polyvinylpyrrolidone is 2:

1.

3. The microneedle acupoint patch according to claim 1, characterized in that, In step S2, the concentration of cinnamon volatile oil in the mixed oil phase is 160–170 mg / mL.

4. The microneedle acupoint patch according to claim 1, characterized in that, In step S3, the emulsifier consists of Tween-80 and anhydrous ethanol.

5. The microneedle acupoint patch according to claim 1, characterized in that, In step S3, the ratio of the mixed oil phase to the emulsifier and water is 6.5–7.5:0.8–1.2:1.8–2.

2.

6. The microneedle acupoint patch according to claim 1, characterized in that, In step S3, the proportion of matrix material in the drug-containing matrix material solution is 70-80%.

7. The microneedle acupoint patch according to claim 1, characterized in that, The backing layer is made of polyvinyl alcohol.

8. The microneedle acupoint patch according to claim 1, characterized in that, The method for preparing the cinnamon volatile oil is to pulverize cinnamon to 20-80 mesh, soak it, extract it with ultrasonic assistance, and distill it at a material-to-liquid ratio of 1:10 for 4-8 hours.

9. The microneedle acupoint patch according to claim 1, characterized in that, The microneedle mold is prepared from polydimethylsiloxane.

10. The use of the microneedle acupoint patch according to any one of claims 1 to 9 in the preparation of a product for treating osteoarthritis.

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