Traditional Chinese medicine volatile oil microneedle preparation for treating acne as well as preparation method and application of traditional Chinese medicine volatile oil microneedle preparation

Abstract

The invention provides a traditional Chinese medicine volatile oil microneedle preparation for treating acne as well as a preparation method and application thereof. The active pharmaceutical ingredient in the microneedle provided by the invention is traditional Chinese medicine volatile oil, the traditional Chinese medicine volatile oil exists in the form of an inclusion compound, and the microneedle is a water-soluble microneedle. Experiments prove that the microneedle disclosed by the invention has the characteristics of stable drug active ingredients, good water solubility, high drug release speed, minimally invasive acne treatment, high safety and effective drug delivery, and the acne treatment effect of the microneedle is remarkably better than that of an existing drug.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a Chinese medicine volatile oil microneedle preparation for treating acne, and a preparation method and application thereof. Background Art

[0002] Acne is a chronic inflammatory disease of the sebaceous glands of the hair follicles. As the second largest skin disease in the world, it directly affects the physical and mental health of adolescents. Acne is mainly caused by excessive secretion of sebum, excessive keratinization of hair follicles, excessive activation of Toll-like receptors by Propionibacterium acnes, and abnormal release of inflammatory cytokines; at the same time, genetics, diet, and stress can also induce or aggravate acne. Therefore, the complexity of the pathogenesis of acne poses a challenge to its treatment. Among the acne treatment options, antibiotics, retinoids, hormones, and photodynamic therapy have certain effects on it, but these methods are often accompanied by poor tolerance and many side effects (such as teratogenicity). Therefore, a safer and more effective treatment method is needed.

[0003] Angelica sinensis is a traditional aromatic Chinese medicine that is used frequently and in large quantities, and has high medicinal and economic value.

[0004] Microneedle patch (MNP) is a new transdermal drug delivery method. Compared with oral and subcutaneous injection, it has unique advantages such as painlessness, good patient compliance, few side effects, and high transdermal delivery efficiency. Summary of the invention

[0005] In order to obtain a safer and more effective drug for treating acne, the inventors of the present invention have conducted a lot of exploration. Through research, it is found that Chinese herbal volatile oil can treat acne, especially Chinese herbal volatile oil with artemisinin as the main component has the effect of treating acne, and further preparing it into the form of inclusion compound can greatly improve the stability and water solubility of volatile oil, and further preparing the inclusion compound of volatile oil into microneedle preparation, effectively delivering the volatile oil inclusion compound for transdermal absorption, greatly improving the effect of treating acne.

[0006] Based on a series of explorations and creations of the present invention, the present invention provides the following technical solutions:

[0007] In one aspect, the present invention provides a microneedle, wherein the active pharmaceutical ingredient in the microneedle is a volatile oil of traditional Chinese medicine, and the volatile oil of traditional Chinese medicine exists in the form of an inclusion compound, and the microneedle is a water-soluble microneedle.

[0008] Preferably, the volatile oil of traditional Chinese medicine is a volatile oil with ligustilide as the main component. Further preferably, the volatile oil with ligustilide as the main component is angelica volatile oil, chuanxiong volatile oil or ligustilide volatile oil.

[0009] One of the innovative points of this technical solution is to make water-soluble microneedles with Chinese medicine volatile oil in the form of inclusion compound. As long as the active ingredient of the medicine is a water-soluble microneedle with inclusion compound of Chinese medicine volatile oil, it belongs to the technical solution of the present invention. As for the specific shape, size, material, etc. of the water-soluble microneedle, as well as the inclusion compound, different choices can be made according to actual needs. The present invention does not list them one by one, nor does it limit them. The technical solution of the present invention is adopted, and the inclusion compound form can improve the stability and water solubility of Chinese medicine volatile oil, and the water-soluble microneedle can improve the transdermal absorption of Chinese medicine volatile oil and improve the therapeutic effect.

[0010] However, as a preferred solution, in order to further improve the stability and water solubility of the volatile oil of traditional Chinese medicine, cyclodextrin is selected as the inclusion material and the volatile oil of traditional Chinese medicine is selected as the inclusion material. The cyclodextrin is preferably β-cyclodextrin, and more preferably hydroxypropyl-β-cyclodextrin.

[0011] As a further preferred technical solution, in order to make the preparation method simple and cost-effective, a grinding method is used to prepare the inclusion compound. In this method, the mass ratio of cyclodextrin and Chinese medicine volatile oil is (8-16):1, preferably (10-15):1 or (10-14):1 or (12-14):1 or (13-14):1 or (13.5-14.5):1, specifically 8:1, 10:1, 12:1, 13:1, 14:1, or 16:1.

[0012] As a further preferred technical solution, based on the above-mentioned feed ratio of cyclodextrin and Chinese medicine volatile oil, the ratio of cyclodextrin and water is further optimized, and the mass ratio of cyclodextrin and water is preferably (1-5):1, more preferably (2.5-1):1 or (1.65-1):1 or (1.65-1.25):1 or (1.6-1.7):1, specifically 5:1, 2.5:1, 1.65:1, 1.67:1, 1.25:1, or 1:1;

[0013] As a further preferred technical solution, based on the above-mentioned feed ratio of cyclodextrin, Chinese medicine volatile oil, and water, the grinding time is further optimized, and the inclusion time is 5-25min, preferably 10-20min or 18-20min, specifically 10min, 15min, 19min or 20min.

[0014] As another preferred technical solution, sodium hyaluronate is selected as the substrate of the microneedle. That is, the main components of the part of the microneedle that penetrates the skin and dissolves in the skin tissue are the Chinese medicine volatile oil inclusion compound and sodium hyaluronate. Further preferably, sodium hyaluronate with a molecular weight of 52kDa is selected. Those skilled in the art can freely choose the shape, size and structure of the microneedle according to actual needs, and this technical solution does not make specific restrictions.

[0015] As a further preferred embodiment, the ratio of the part of the Chinese medicine volatile oil inclusion compound and sodium hyaluronate in the above-mentioned microneedle that penetrates the skin and dissolves in the skin tissue is selected as: 100mg-300mg of Chinese medicine volatile oil inclusion compound: 250mg of sodium hyaluronate, and more specifically 100mg of Chinese medicine volatile oil inclusion compound: 250mg of sodium hyaluronate, 200mg of Chinese medicine volatile oil inclusion compound: 250mg of sodium hyaluronate or 300mg of Chinese medicine volatile oil inclusion compound: 250mg of sodium hyaluronate.

[0016] Correspondingly, the part of the above-mentioned microneedle that penetrates the skin and dissolves in the skin tissue is made of a solution, which consists of sodium hyaluronate, a Chinese medicine volatile oil inclusion compound and water; further preferably, the composition and ratio of the solution are: 100mg-300mg of a Chinese medicine volatile oil inclusion compound and 250mg of sodium hyaluronate are added to every 1ml of water; more specifically, 100mg, 200mg or 300mg of a Chinese medicine volatile oil inclusion compound and 250mg of sodium hyaluronate are added to every 1ml of water.

[0017] As another preferred embodiment, the microneedle structure of the present invention is composed of a needle tip and a base connected to the non-tip end of the needle tip. In this embodiment, the main components of the needle tip are a Chinese medicine volatile oil inclusion compound and sodium hyaluronate, and further preferably, sodium hyaluronate with a molecular weight of 52kDa is selected. The main components of the base are sodium hyaluronate with a molecular weight of 52kDa, dextran, and sodium hyaluronate with a molecular weight of 200-400kDa. Preferably, the molecular weight of dextran is 40000.

[0018] Preferably, the ratio of the Chinese medicine volatile oil inclusion compound and sodium hyaluronate in the needle tip is: 100mg-300mg Chinese medicine volatile oil inclusion compound: 250mg sodium hyaluronate, and more specifically 100mg Chinese medicine volatile oil inclusion compound: 250mg sodium hyaluronate, 200mg Chinese medicine volatile oil inclusion compound: 250mg sodium hyaluronate or 300mg Chinese medicine volatile oil inclusion compound: 250mg sodium hyaluronate.

[0019] Further preferably, among the main components of the base, the mass ratio of sodium hyaluronate with a molecular weight of 52 kDa, dextran and sodium hyaluronate with a molecular weight of 200-400 kDa is 1:1:1.

[0020] Correspondingly preferably, the needle tip is made of a solution consisting of sodium hyaluronate, Chinese herbal volatile oil and water; further preferably, the composition and ratio of the solution are: 100mg-300mg of Chinese herbal volatile oil inclusion compound and 250mg of sodium hyaluronate are added to every 1ml of water; more specifically, 100mg, 200mg or 300mg of Chinese herbal volatile oil inclusion compound and 250mg of sodium hyaluronate are added to every 1ml of water.

[0021] Correspondingly preferably, the above-mentioned base is made of the following solution: 100 mg of sodium hyaluronate with a molecular weight of 52 kDa, 100 mg of dextran, and 100 mg of sodium hyaluronate with a molecular weight of 200-400 kDa are added to every 1 ml of water.

[0022] As a preferred technical solution, in any of the above-mentioned microneedles, the drug loading of a single microneedle is 0.09 μg of Chinese medicine volatile oil / microneedle to 0.39 μg of Chinese medicine volatile oil / microneedle, specifically 0.09 μg of Chinese medicine volatile oil / microneedle, 0.21 μg of Chinese medicine volatile oil / microneedle, or 0.39 μg of Chinese medicine volatile oil / microneedle.

[0023] As a preferred technical solution, in any of the above-mentioned microneedles, the mechanical strength of the microneedle is at least 90N.

[0024] As a preferred technical solution, in any of the above-mentioned microneedles, the microneedle can penetrate the stratum corneum, enter the epidermis and even the dermis.

[0025] As a preferred technical solution, in the above-mentioned microneedle having a needle tip and a base, the ratio of the solution for preparing the needle tip to the solution for preparing the base is 60 mg needle tip: 140 mg base.

[0026] As a preferred technical solution, the amount of solution used to prepare a single microneedle tip is 60 mg, and the amount of solution used to prepare a single microneedle base is 140 mg.

[0027] As a preferred technical solution, in the above-mentioned microneedle with a needle tip and a base, the top of the microneedle tip is a quadrangular pyramid with a height of 600μm and a needle tip width of 300μm, the bottom of the needle tip is a regular quadrangular prism with a height of 600μm, and the total length of the microneedle tip is 1200μm.

[0028] As a preferred technical solution, any one of the above-mentioned microneedles is a microneedle for treating acne.

[0029] The microneedle formed by any combination of any of the above preferred technical solutions is also a technical solution requested for protection by the present invention.

[0030] The present invention also provides a microneedle patch, comprising an array consisting of a plurality of the above-mentioned single microneedles.

[0031] As a preferred technical solution, the content of Chinese medicine volatile oil in each microneedle patch is 12.96±2.67μg-56.00±4.08μg, specifically 12.96±2.67μg, 30.45±0.62μg or 56.00±4.08μg.

[0032] As a further preferred technical solution, the microneedle array is a 12×12 microneedle array, the tip of each microneedle is 1200 μm long, and the distance between each needle is 600 μm; the top of the microneedle tip is a quadrangular pyramid with a height of 600 μm, the tip width is 300 μm, and the bottom of the tip is a regular quadrangular prism with a height of 600 μm.

[0033] The invention provides a method for preparing a Chinese medicine volatile oil inclusion compound. The inclusion compound is prepared by a grinding method. In the method, the feeding mass ratio of cyclodextrin to the Chinese medicine volatile oil is (8-16):1, preferably (10-15):1 or (10-14):1 or (12-14):1 or (13-14):1 or (13.5-14.5):1, specifically 8:1, 10:1, 12:1, 13:1, 14:1, or 16:1.

[0034] As a further preferred technical solution, based on the above-mentioned feed ratio of cyclodextrin and Chinese medicine volatile oil, the ratio of cyclodextrin and water is further optimized, and the mass ratio of cyclodextrin and water is preferably (1-5):1, more preferably (2.5-1):1 or (1.65-1):1 or (1.65-1.25):1 or (1.6-1.7):1, specifically 5:1, 2.5:1, 1.65:1, 1.67:1, 1.25:1, or 1:1;

[0035] As a further preferred technical solution, based on the above-mentioned feed ratio of cyclodextrin, Chinese medicine volatile oil and water, the grinding time is further optimized, and the inclusion time is 5-25min, preferably 10-20min or 18-20min, specifically 10min, 15min, 19min or 20min.

[0036] Preferably, the cyclodextrin is β-cyclodextrin, and more preferably hydroxypropyl-β-cyclodextrin.

[0037] The present invention provides an inclusion compound of a volatile oil of a traditional Chinese medicine, wherein cyclodextrin is selected as an inclusion material and the volatile oil of the traditional Chinese medicine is selected as a material to be included, wherein the cyclodextrin is preferably β-cyclodextrin, and more preferably hydroxypropyl-β-cyclodextrin.

[0038] As a preferred embodiment, the inclusion compound is prepared by the above-mentioned grinding method.

[0039] The present invention provides a method for preparing a microneedle, comprising the following steps:

[0040] Filling any of the above solutions for preparing microneedle tips into a mold, and centrifuging until the solution fills the mold tip and flattens it;

[0041] Then, any of the above solutions for preparing the microneedle base is added into the mold and centrifuged until the solution fills the mold and flattens it;

[0042] dry.

[0043] The microneedle preparation method of the present invention may also include any of the above-mentioned methods for preparing the inclusion compound.

[0044] The present invention also provides a technical solution, which is the use of any of the above-mentioned microneedles or microneedle patches in the preparation of a drug for treating acne.

[0045] In any of the above-mentioned technical solutions (microneedles, inclusion compounds, microneedle preparation methods, inclusion compound preparation methods, and application methods), Chinese herbal volatile oil refers to volatile oil components contained in Chinese herbal medicine with ligustilide as the main component.

[0046] Among them, taking ligustilide as the main component means that the content of ligustilide is the main component. The following situations all belong to the Chinese medicine volatile oil with ligustilide as the main component: the mass percentage of ligustilide in the volatile oil is more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 79%, more than 80%, more than 85%, more than 90%, more than 95% or 100%; or the mass percentage of ligustilide in the volatile oil is 75%-85% or 78%-82%; or the mass percentage of ligustilide in the volatile oil is 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0047] As specific examples, the volatile oil containing ligustilide as a main component is angelica volatile oil, chuanxiong rhizome volatile oil or ligustilide volatile oil.

[0048] The volatile oils of Chinese medicine can be extracted by yourself or purchased directly.

[0049] As a further specific example, the components and contents of Angelica dahurica volatile oil are shown in Table 1.

[0050] The volatile oil of Angelica sinensis can be extracted by the method disclosed in the prior art. As an example of the present invention, it can be obtained by steam distillation.

[0051] definition:

[0052] AEO: Essential oil from Angelica sinensis.

[0053] HPCD: Hydroxypropyl-β-cyclodextrin.

[0054] AEO@HPCD: Angelica volatile oil inclusion complex.

[0055] MNs: microneedles.

[0056] AEO@HPCD MNs: Angelica volatile oil inclusion complex microneedles.

[0057] HA: Sodium hyaluronate.

[0058] Experiments have shown that the microneedles of the present invention have stable active drug ingredients, good water solubility, and fast drug release. They are minimally invasive, highly safe, and can effectively deliver drugs for acne, and their effect in treating acne is significantly better than that of existing drugs.

[0059] The present invention uses hydroxypropyl-β-cyclodextrin (HP-β-CD) to prepare inclusion compounds of Chinese herbal volatile oils, thereby enhancing the water solubility and stability of Chinese herbal volatile oils. The inclusion compounds are then made into microneedles. The microneedles prepared by the present invention can effectively pierce the stratum corneum of the skin to deliver the inclusion compounds into the acne site, and are painless, minimally invasive, highly safe, and effectively deliver drugs, thereby ensuring the pharmacological activity of Chinese herbal volatile oils and achieving the purpose of effectively treating acne.

[0060] Chinese medicine volatile oil is composed of compounds with a molecular weight of less than 300, which can easily penetrate the stratum corneum to exert its medicinal effect. However, after the inclusion of Chinese medicine volatile oil, the molecular weight increases and it is not easy to penetrate the stratum corneum. Through microneedle technology, the inclusion compound is loaded onto the microneedle, and the mechanical force suitable for the microneedle is used to penetrate the stratum corneum, enter the dermis, release the inclusion compound and Chinese medicine volatile oil, and thus exert the pharmacological effect of Chinese medicine volatile oil. Combined with the characteristics of acne, the present invention prepares water-soluble microneedles, optimizes the microneedle formula, and adopts a mold method and a two-step centrifugation method to achieve simple and efficient preparation of microneedles. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 These are the results of stability investigation on AEO@HPCD.

[0062] Figure 2 This is the appearance morphology of microneedle AEO@HPCD MNs.

[0063] Figure 3 Mechanical force of microneedle AEO@HPCD MNs.

[0064] Figure 4 HE staining after microneedles were applied to mouse skin.

[0065] Figure 5 For the safety assessment of microneedles.

[0066] Figure 6 Cumulative release curve of AEO in microneedles.

[0067] Figure 7 This is a graph showing the changes in acne volume in each group of mice over 7 days in the acne model.

[0068] Figure 8 This is the result graph of acne volume of each group of mice on the 7th day in the acne model.

[0069] Fig. 9 The morphological changes of acne in mice in each group within 7 days.

[0070] Fig.10 It is the bar graph of the mechanical force of microneedles in each group.

[0071] Fig.11 It is the porosity curve of each group of microneedles.

[0072] Fig.12 It is a bar graph of the drug loading of microneedles in each group.

[0073] Fig.13 is the encapsulation rate of AEO in the inclusion complex at various ratios of HPCD:AEO (g / g).

[0074] Fig.14 is the encapsulation rate of AEO in the inclusion complex at various ratios of HPCD:Water (g / g).

[0075] Fig.15 is the encapsulation efficiency of AEO in the inclusion complex under the grinding time. DETAILED DESCRIPTION

[0076] The following are some specific embodiments of the present invention, wherein the technical means or reagents used are conventional methods and reagents known in the art unless otherwise specified.

[0077] Embodiment 1 Angelica dahurica volatile oil

[0078] Angelica volatile oil was obtained by steam distillation; Angelica medicinal materials were purchased from Chengdu Chengshitong Trading Co., Ltd., 200 kg of Angelica was added with appropriate amount of water, soaked for 8-12 hours, loaded into a Chinese extraction device, steam was introduced, and extracted for 9 hours; Angelica volatile oil was collected, and dehydrated by adding appropriate amount of anhydrous sodium sulfate; the volatile oil was put into a brown glass bottle and stored at 4°C. The GC-MS analysis results are shown in Table 1.

[0079] Table 1 GC-MS analysis results of angelica volatile oil

[0080]

[0081]

[0082] Example 2 Preparation method of angelica volatile oil inclusion compound (AEO@HPCD)

[0083] The preparation method of AEO@HPCD was obtained by grinding method through single factor investigation and Box-Behnken design.

[0084] The process flow of preparing AEO@HPCD by grinding method is as follows: water is added at a mass ratio of HPCD: water = 1.65. After HPCD is dissolved, AEO is added at a mass ratio of HPCD: AEO = 14, and ground for 19 minutes in a grinder (25°C, 60HZ). The mixture is centrifuged, and the unencapsulated AEO is washed away with 80% ethanol. The mixture is freeze-dried to obtain a dry and loose inclusion complex AEO@HPCD powder.

[0085] Example 3 Microneedles

[0086] AEO@HPCD MNs are composed of AEO@HPCD, HA (52 kDa), HA (200-400 kDa), and dextran 40 (molecular weight of 40,000).

[0087] The matrix ratio of AEO@HPCD MNs tip is: 300 mg AEO@HPCD and 250 mg HA (52 kDa) are added to 1 ml water;

[0088] The matrix ratio of AEO@HPCD MNs base is: 100 mg HA (52 kDa), 100 mg dextran 40, and 100 mg HA (200-400 kDa) are added to 1 ml of water;

[0089] The needle tip matrix and the base matrix were mixed according to the prescription ratio, and 60 mg of the needle tip matrix and 140 mg of the base matrix were taken to prepare AEO@HPCD MNs.

[0090] Microneedles were prepared using the microneedle mold method and the two-step centrifugation method.

[0091] The microneedle mold is a 12×12 microneedle array, each microneedle is 1200μm long, and the distance between each needle is 600μm; the tip width is 300μm, the bottom is a regular quadrangular prism, the height is 600μm, and the top is a quadrangular pyramid, the height is 600μm. The AEO@HPCD MNs prescription is obtained according to the AEO content, the mechanical strength of the microneedle, the penetration depth, etc. The microneedle preparation method is as follows: ① Add 300mg AEO@HPCD and 250mg sodium hyaluronate (52kDa) to 1ml water and mix well to obtain a microneedle tip matrix solution. ②100 mg HA (52 kDa), 100 mg dextran 40 and 100 mg HA (200-400 kDa) were added to 1 ml of water and mixed to obtain a microneedle base matrix solution; ③ About 60 mg of the needle tip matrix solution was added to the microneedle mold, centrifuged at 4000 rpm for 3 min at low temperature (4°C), and the mold direction was changed (rotated 180°) and centrifuged 6 times until the needle tip matrix solution filled the mold needle tip and was flattened; ④ About 140 mg of the microneedle base matrix solution was added to the microneedle groove in the mold, centrifuged at 3000 rpm for 3 min at low temperature, and the mold direction was changed (rotated 180°) and centrifuged 4 times until the base matrix was flattened in the mold; dried at room temperature for 48 h to obtain microneedle AEO@HPCD MNs.

[0092] Example 4 AEO@HPCD improves the stability and water solubility of AEO

[0093] The high temperature accelerated test was conducted. The samples were spread into a thickness of ≤5 mm and placed at 60°C. The contents of ligustilide in AEO, the physical mixture of HPCD and AEO (HPCD+AEO) and AEO@HPCD were determined on the 0th, 5th and 10th day, respectively. The relative retention rate of ligustilide was calculated compared with that on the 0th day. The results are shown in Table 1. Figure 1 Compared with AEO@HPCD, the relative retention rates of ligustilide in AEO and HPCD+AEO were significantly reduced on the 5th day (P=0.0046, P=0.0152) and the 10th day (P=0.0168, P=0.0008), which proved that AEO@HPCD improved the stability of AEO. Figure 1 .

[0094] The phase solubility method was adopted. An appropriate amount of HPCD was weighed and distilled water was added to prepare 5 mL of solutions with concentrations of 0, 10, 20, 30, 40, and 50 mmol / L, respectively. Excess volatile oil of Angelica sinensis was added and shaken at 37°C and 120 rpm in a constant temperature oscillator for 48 h. After the solution was balanced, it was centrifuged at 12,000 rpm for 10 min at room temperature. The supernatant was taken, diluted, filtered, and the filtrate was taken for HPLC determination. The phase solubility curve was drawn with HPCD concentration (mmol / L) as the abscissa and ligustilide concentration (mmol / L) as the ordinate. Linear regression was performed on the curve to obtain the regression equation. According to the theory proposed by Higuchi and Connors, the solubilization multiple was calculated, and the results are shown in Table 2. Within the experimental concentration range, the maximum solubilization multiple of ligustilide can reach 22.66 times, indicating that HPCD can increase the water solubility of ligustilide, thereby indirectly reflecting that HPCD can increase the water solubility of AEO.

[0095] Table 2 Solubilization multiples of ligustilide

[0096]

[0097] Example 5 Obtaining a microneedle AEO@HPCD MNs preparation that can effectively penetrate the stratum corneum of the skin and is safe to use

[0098] The appearance and morphology of the microneedle AEO@HPCD MNs prepared by the mold method and the two-step centrifugation method are shown in Figure 2 The mechanical force of the microneedle AEO@HPCD MNs reaches more than 90N, which meets the mechanical properties when penetrating the stratum corneum of the skin. Figure 3 .

[0099] AEO@HPCD MNs can effectively deliver AEO@HPCD to subcutaneous tissue: After microneedle AEO@HPCD MNs were inserted into the back skin of mice using the skin section HE staining method, the skin tissue was separated and HE staining was performed to observe the microneedle penetration. The results are shown in Figure 4 .from Figure 4 It was observed that the microneedles could penetrate the stratum corneum, epidermis and reach the dermis, with the penetration depth reaching 110 μm, indicating that the microneedles could penetrate the stratum corneum, enter the epidermis and even the dermis tissue.

[0100] Safety of AEO@HPCD MNs application: The transepidermal water loss value (TEWL) was used to reflect the integrity of the skin barrier function and to investigate the safety of AEO@HPCD MNs. Figure 5. In the experiment, the TEWL value of normal SD rat skin was (2.88±0.32) g / m2h. After AEO@HPCD MNs were inserted into the skin for 5 minutes, obvious micropores could be observed on the skin surface, arranged in a 12×12 array, and the skin TEWL value reached a maximum value of (10.22±1.91) g / m2h, indicating that the prepared microneedles had good mechanical strength and high skin porosity, and could effectively pierce the stratum corneum barrier and form micropores on the skin surface. At 20 minutes, there were still micropores on the skin surface, but the micropores began to decrease, and the TEWL value decreased to (6.53±1.08) g / m2h. After that, with the increase of time, the skin micropores gradually disappeared, the skin TEWL value also gradually decreased, and the skin barrier function gradually recovered. 180 minutes after microneedle insertion, the TEWL value of the skin recovered to (2.98±0.46) g / m2h, which is not much different from normal skin, indicating that the micropores generated by the microneedles have basically recovered completely at this time, indicating that AEO@HPCD MNs have good safety after use. Figure 5 .

[0101] Microneedle AEO@HPCD MNs can release AEO quickly: The cumulative release of AEO in the microneedles was determined by in vitro release experiments. A 40mmol / L HPCD solution was used as the receiving solution. The release receiving solution was taken at different time points to determine the AEO content and calculate the cumulative release of AEO. The results are shown in Figure 6 At 0.5 h, the cumulative release of AEO was about 20%, at 2 h about 45%, and at 6 h about 75%. At 12 h, the cumulative release of AEO in the microneedles was about 95%, and AEO was completely released, which was consistent with the Higuchi equation (Qt = 15.343t 1 / 2 +12.532,R 2 =0.8873), indicating that AEO in the microneedles has the characteristic of rapid release.

[0102] Microneedle AEO@HPCD MNs can treat acne more effectively: A mouse acne model was established, and the commercially available adapalene gel (ADA gel) was used as a positive control. The Model group, ADA gel group, AEO (%) group, AEO@HPCD group, blank microneedle (Blank MNs) group, and microneedle (AEO@HPCD MNs) group were set up. The day when the mouse skin bulges and bulges formed acne was recorded as the first day. The acne volume on the first day was taken as the relative value, and the percentage of the acne volume on each subsequent day relative to the volume on the first day was calculated ( Figure 7 ), and the relative acne volume on the seventh day ( Figure 8 ), the changes in the appearance and volume of acne in each group of mice were shown in Fig. 9. On the first day, the size of acne in each group was similar. In the Model group, the volume of acne increased throughout the process, and on the 7th day, it was 149.78% of the initial volume. Compared with the Model group, the volume of acne in the drug-treated groups on the second day no longer increased but showed a decreasing trend, and there was a statistical difference (P<0.001). The volume of acne in the AEO@HPCD MNs group was significantly smaller after three days of drug administration, and at the end of the experiment, the acne was so small that it was about to disappear; the volume of acne in other drug-treated groups was significantly smaller on the 5th or 7th day. Among them, the volume of acne in the Blank MNs group was 85.89% of the initial volume on the 7th day, which was statistically different from the Model group (P<0.001). This may be due to the micro-channels formed by the microneedles penetrating the skin, which destroyed the anaerobic microenvironment of Propionibacterium acnes (P.acnes), affecting the growth of P.acnes. The acne volumes of the ADA gel group, AEO (10%) group, AEO@HPCD group, and AEO@HPCD MNs group on the 7th day were 67.35%, 75.18%, 81.68%, and 34.86% of the initial values, respectively, which were statistically different from the Model group (P<0.001), indicating that the drug-treated groups all had a certain therapeutic effect on the mouse acne model. However, the relative volume of acne in the AEO@HPCD MNs group was significantly smaller than that in the ADA gel group, AEO (10%) group, AEO@HPCD group, and Blank MNs group, indicating that the inhibitory effect of AEO@HPCD MNs on acne growth was better than that of the commercially available preparations ADA gel, AEO (%), AEO@HPCD, and Blank MNs, indicating that AEO@HPCD MNs has obvious advantages in the treatment of mouse acne models.

[0103] Example 6 Microneedles with different formulations

[0104] 50mg-400mg of inclusion compound AEO@HPCD was selected for the experiment. As a result, when 50mg of inclusion compound was added, the drug loading of the microneedle was too low, and when 400mg of inclusion compound was added, the microneedle tip matrix solution was unevenly dispersed. Therefore, microneedles were prepared with inclusion compounds added in the range of 100mg-300mg. The microneedle preparation method was the same as in Example 3, except that the amount of inclusion compound added was different, and the rest remained unchanged. Determine the relevant parameters. The determination of microneedle mechanical strength, microneedle porosity, and microneedle drug loading are all conventional detection methods in the field. The drug loading is defined as the mass of AEO contained in a microneedle.

[0105] Microneedle mechanical force measurement: Blank microneedles (Blank MNs), (100mg) AEO@HPCD MNs, (200mg) AEO@HPCD MNs, and (300mg) AEO@HPCD MNs all have mechanical forces above 90N, which is sufficient to penetrate the stratum corneum of the skin. Fig.10.

[0106] The porosity of the microneedles was measured by using a thin film to simulate the thickness of human skin to examine the penetration depth. Results: The porosity of blank microneedles (Blank MNs), (100mg) AEO@HPCD MNs, (200mg) AEO@HPCD MNs, and (300mg) AEO@HPCD MNs increased with the increase of inclusion complexes, and some needles of (300mg) AEO@HPCD MNs could penetrate to a depth of 600μm. Results are shown in Fig.11 .

[0107] The drug loading of the microneedles was measured. The results showed that the drug loading in the microneedles of (100 mg) AEO@HPCD MNs, (200 mg) AEO@HPCD MNs, and (300 mg) AEO@HPCD MNs increased with the increase of the inclusion complex. When (300 mg) AEO@HPCD MNs was used, the drug loading of the microneedles could reach the effective dose for treating acne. The results are shown in Fig.12 .

[0108] Example 7 Different processes for preparing inclusion compounds

[0109] 1. Process flow of preparing inclusion complex by grinding method: HPCD is added with a certain amount of water, dissolved, and then a certain amount of AEO is added, ground, centrifuged, and unincluded AEO is washed away with 80% ethanol, and freeze-dried to obtain inclusion complex powder.

[0110] 2. Single factor method for initial screening of feed-oil ratio (HPCD:AEO), feed-water ratio (HPCD:Water) and grinding time range

[0111] Screening of feed-oil ratio (HPCD: AEO): Under the conditions of HPCD: Water (g / g) = 1.67:1 and grinding time of 15 min, the inclusion complex was prepared according to the process flow with the ratio of HPCD: AEO (g / g) of 8:1, 10:1, 12:1, 14:1, and 16:1. The AEO content in the inclusion complex was determined and the AEO encapsulation rate was calculated. The results are shown in Fig.13 When the HPCD:AEO (g / g) ratio was 14:1, the AEO encapsulation efficiency was the highest.

[0112] Screening of material-water ratio (HPCD: Water): Under the conditions of HPCD: AEO (g / g) = 10:1 and grinding time of 15 min, the inclusion complex was prepared according to the process flow with HPCD: Water (g / g) ratios of 5:1, 2.5:1, 1.67:1, 1.25:1, and 1:1. The AEO content in the inclusion complex was determined and the AEO encapsulation rate was calculated. The results are shown in Fig.14When the HPCD:Water (g / g) ratio was 1.67:1, the AEO encapsulation efficiency was the highest.

[0113] Screening of milling time (MT): Under the conditions of HPCD:AEO (g / g) = 10:1, HPCD:Water (g / g) = 1.67:1, the milling time was 5, 10, 15, 20, and 25 min, respectively, and the inclusion complex was prepared according to the process flow. The AEO content in the inclusion complex was determined and the AEO encapsulation rate was calculated. The results are shown in Fig.15 When the grinding time was 15 min, the AEO encapsulation efficiency was the highest.

[0114] Based on the above results, the single factor method obtained three level ranges of three factors [feed-to-oil ratio (HPCD:AEO), feed-to-water ratio (HPCD:Water) and grinding time], see Table 3.

[0115] Table 3 Conditions of 3 factors and 3 levels of inclusion compound grinding method

[0116] Factors Levels AEO:HPCD(g / g) 1:10,1:12,1:14 MT(min) 10,15,20 HPCD:Water(g / g) 1.67:1,1.25:1,1:1

[0117] 3. Determination of inclusion compound preparation conditions by response surface methodology (BoX-Behnken)

[0118] The experimental design table was obtained by using the software "Design Expert 8.0.6": the comprehensive score value was used as an indicator. Comprehensive score value = 0.7 × encapsulation efficiency + 0.3 × yield. The comprehensive score results under the experimental conditions are shown in Table 4.

[0119] Table 4 Experimental design table and response values

[0120]

[0121]

[0122] The results were fitted to obtain the optimal process conditions: According to the results in Table 4, the comprehensive score was used as the dependent variable, and the binomial equation was fitted for the feed-oil ratio (HPCD:AEO), feed-water ratio (HPCD:Water) and grinding time. The results are shown in Table 5.

[0123] Table 5 ANOVA analysis results of quadratic polynomial model

[0124]

[0125] There was a significant difference in the model (P = 0.004), and there was no significant difference in the Lack of Fit (P = 0.1061), indicating that the model fit was good. After analysis, the multivariate binomial regression equation was obtained: Comprehensive Score = 61.14 + 4.84A + 3.87B + 3.79C - 0.92AB + 3.64AC + 1.30BC + 1.75A^2 + 1.11B^2 + 2.13C^2, R 2 =0.9219, R 2 Adj =0.8215.

[0126] The maximum value of the comprehensive score was set to obtain the optimal conditions for feed-oil ratio (HPCD:AEO), feed-water ratio (HPCD:Water) and grinding time. The results are shown in Table 6.

[0127] Table 6 Optimal parameter conditions

[0128]

[0129] According to the actual situation, the parameter conditions were adjusted to HPCD:AEO=14, MT=19, HPCD:Water=1.65. Under this condition, three verification experiments were carried out and the results are shown in Table 7.

[0130] Table 7 Verification experimental results

[0131]

[0132] The comprehensive score value after adjusting the parameter conditions deviated from the model prediction value by 1.38%, and the experimental value was close to the predicted value, indicating that the experimental conditions can be adjusted to HPCD:AEO=14, MT=19, and HPCD:Water=1.65.

[0133] Based on the above results, the parameters and process flow of the grinding method for preparing the inclusion complex are as follows: water is added at a ratio of HPCD:Water=1.65. After HPCD is dissolved, AEO is added at a ratio of HPCD:AEO=14, and the mixture is ground in a grinder (25°C, 60HZ) for 19 minutes. The mixture is centrifuged, unencapsulated AEO is washed off with 80% ethanol, and the mixture is freeze-dried to obtain a dry and loose inclusion complex powder.

[0134] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A microneedle, characterized in that: The active ingredient of the medicine in the microneedle is volatile oil of traditional Chinese medicine, which exists in the form of inclusion compound. The microneedle is a water-soluble microneedle.

2. The microneedle according to claim 1, characterized in that: In the inclusion compound, the included material is the volatile oil of traditional Chinese medicine, and the inclusion material is cyclodextrin, preferably β-cyclodextrin, and more preferably hydroxypropyl-β-cyclodextrin.

3. The microneedle according to claim 1 or 2, characterized in that: The inclusion compound is prepared by a grinding method; Preferably, the mass ratio of cyclodextrin to Chinese herbal volatile oil is (8-16):1, preferably (10-15):1 or (10-14):1 or (12-14):1 or (13-14):1 or (13.5-14.5):1, specifically 8:1, 10:1, 12:1, 13:1, 14:1, or 16:1; Further preferably, the mass ratio of cyclodextrin to water is (1-5):1, preferably (2.5-1):1 or (1.65-1):1 or (1.65-1.25):1 or (1.6-1.7):1, specifically 5:1, 2.5:1, 1.65:1, 1.67:1, 1.25:1, or 1:1; Further preferably, in the method for preparing the inclusion compound, the inclusion time is 5-25 min, preferably 10-20 min or 18-20 min, specifically 10 min, 15 min, 19 min or 20 min.

4. The microneedle according to any one of claims 1 to 3, characterized in that: The main components of the part of the microneedle that penetrates the skin and dissolves in the skin tissue are the Chinese medicine volatile oil inclusion compound and sodium hyaluronate; Preferably, the ratio of the Chinese medicine volatile oil inclusion compound and sodium hyaluronate is 100mg-300mg of the Chinese medicine volatile oil inclusion compound: 250mg of sodium hyaluronate, and more specifically, 100mg of the Chinese medicine volatile oil inclusion compound: 250mg of sodium hyaluronate, 200mg of the Chinese medicine volatile oil inclusion compound: 250mg of sodium hyaluronate, or 300mg of the Chinese medicine volatile oil inclusion compound: 250mg of sodium hyaluronate; Further preferably, the part of the microneedle that penetrates the skin and dissolves in the skin tissue is made of a solution, and the composition and proportion of the solution are: 100 mg-300 mg of the Chinese medicine volatile oil inclusion compound and 250 mg of sodium hyaluronate are added to every 1 ml of water; more specifically, 100 mg, 200 mg or 300 mg of the Chinese medicine volatile oil inclusion compound and 250 mg of sodium hyaluronate are added to every 1 ml of water; More preferably, the molecular weight of sodium hyaluronate is 52 kDa.

5. The microneedle according to any one of claims 1 to 4, characterized in that: The microneedle structure comprises a needle tip and a base connected to a non-tip end of the needle tip, wherein the needle tip is the portion of the microneedle according to claim 4 that penetrates the skin and dissolves in the skin tissue; Preferably, the main components of the base are sodium hyaluronate with a molecular weight of 52 kDa, dextran and sodium hyaluronate with a molecular weight of 200-400 kDa; Further preferably, among the main components of the base, the mass ratio of sodium hyaluronate with a molecular weight of 52 kDa, dextran and sodium hyaluronate with a molecular weight of 200-400 kDa is 1:1:1; Still further preferably, the base is made of the following solution: 100 mg of sodium hyaluronate with a molecular weight of 52 kDa, 100 mg of dextran, and 100 mg of sodium hyaluronate with a molecular weight of 200-400 kDa are added to every 1 ml of water; More preferably, the molecular weight of dextran is 40,000.

6. The microneedle according to any one of claims 1 to 5, characterized in that: The drug loading of a single microneedle is 0.09 μg of Chinese medicine volatile oil / microneedle to 0.39 μg of Chinese medicine volatile oil / microneedle, specifically 0.09 μg of Chinese medicine volatile oil / microneedle, 0.21 μg of Chinese medicine volatile oil / microneedle, or 0.39 μg of Chinese medicine volatile oil / microneedle; and / or, the mechanical strength of the microneedle is at least 90N; And / or, the ratio of the solution for preparing the needle tip to the solution for preparing the base is 60 mg needle tip: 140 mg base; And / or, the amount of solution used to prepare a single microneedle tip is 60 mg, and the amount of solution used to prepare a single microneedle base is 140 mg.

7. The microneedle according to any one of claims 1 to 6, characterized in that: The microneedles are used to treat acne; And / or, the top of the needle tip of the microneedle is in the shape of a quadrangular pyramid with a height of 600 μm, the needle tip width is 300 μm, and the bottom of the needle tip is in the shape of a regular quadrangular prism with a height of 600 μm. And / or, the microneedle is prepared according to the method of claim 10; Preferably, the Chinese herbal volatile oil is a volatile oil with ligustilide as the main component. Further preferably, the volatile oil with ligustilide as the main component is Angelica sinensis volatile oil, Ligusticum chuanxiong volatile oil or Ligusticum chuanxiong volatile oil.

8. A microneedle patch comprising at least one microneedle according to any one of claims 1 to 7, or comprising an array consisting of several microneedles according to any one of claims 1 to 7; Preferably, the content of volatile oil of traditional Chinese medicine in each microneedle patch is 12.96±2.67 μg-56.00±4.08 μg, specifically 12.96±2.67 μg, 30.45±0.62 μg or 56.00±4.08 μg.

9. An inclusion compound, wherein the inclusion material is a volatile oil of traditional Chinese medicine and the inclusion material is cyclodextrin; The inclusion compound is prepared by a grinding method; Preferably, the mass ratio of cyclodextrin to Chinese herbal volatile oil is (8-16):1, preferably (10-15):1 or (10-14):1 or (12-14):1 or (13-14):1 or (13.5-14.5):1, specifically 8:1, 10:1, 12:1, 13:1, 14:1, or 16:1; Further preferably, the mass ratio of cyclodextrin to water is (1-5):1, preferably (2.5-1):1 or (1.65-1):1 or (1.65-1.25):1 or (1.6-1.7):1, specifically 5:1, 2.5:1, 1.65:1, 1.67:1, 1.25:1, or 1:1; Further preferably, in the method for preparing the inclusion compound, the inclusion time is 5-25 min, preferably 10-20 min or 18-20 min, specifically 10 min, 15 min, 19 min or 20 min; More preferably, the cyclodextrin is β-cyclodextrin, and more preferably hydroxypropyl-β-cyclodextrin; More preferably, the Chinese medicine volatile oil is a volatile oil with ligustilide as a main component, and even more preferably, the volatile oil with ligustilide as a main component is Angelica sinensis volatile oil, Chuanxiong volatile oil or Ligusticum chuanxiong volatile oil.

10. The method for preparing the microneedle according to any one of claims 1 to 7, comprising the following steps: Filling the solution for preparing the microneedle tip according to any one of claims 1 to 7 into a mold, and centrifuging until the solution fills the mold tip and flattens it; Then, the solution for preparing the microneedle base according to any one of claims 1 to 7 is added into the mold, and centrifuged until the solution fills the mold and is flattened; dry; Use of the microneedle or microneedle patch according to any one of claims 1 to 8 in the preparation of a drug for treating acne.

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