Preparation of artemether-cyclodextrin inclusion compound and application of artemether-cyclodextrin inclusion compound in shampoo

Abstract

The invention discloses a preparation method of a water-soluble artemether-cyclodextrin inclusion compound and application of the water-soluble artemether-cyclodextrin inclusion compound in shampoo. The artemether-cyclodextrin inclusion compound comprises artemether, cyclodextrin and a complex, and the water solubility and quick release property of the inclusion compound are improved. The preparation method of the artemether-cyclodextrin inclusion compound comprises the following steps: respectively preparing an artemether ethanol solution and a cyclodextrin aqueous solution, mixing the two solutions, carrying out centrifugation and other treatments to form a mixed solution containing the artemether-cyclodextrin inclusion compound, and carrying out evaporation and freeze drying on the mixed solution to obtain powder of the artemether-cyclodextrin inclusion compound. Compared with monomer artemether, the clathrate has the advantages of high water solubility, quick release and the like. The inclusion compound can be used for preparing shampoo, the concentration of the inclusion compound in shampoo is 1-50 g / L, and the inclusion compound can be used for treating scalp seborrheic dermatitis and other diseases caused by pityrosporum orbiculare infection.

Description

Technical Field

[0001] The invention relates to the field of medicines, and in particular to a method for preparing a water-soluble artemether-cyclodextrin inclusion compound and application of the same in shampoo. Background Art

[0002] Artemether is a monomer compound of Artemisia annua. The raw material has low solubility in aqueous solution and relatively slow drug release rate. However, artemether loaded with cyclodextrin nanoparticles can significantly increase the drug release rate. Domestic scholars have studied artemether in cyclodextrin nanosponges, and the cumulative release of artemether can reach 85% in 5 minutes, which has a significant rapid release effect (Study on cyclodextrin nanosponges and their artemether drug delivery system, postgraduate student Xu Jianghui, tutor Zhang Jiwen, Anhui University of Chinese Medicine, 2017 master's degree thesis). However, cyclodextrin nanosponges-artemether inclusion complexes are polymers of multiple cyclodextrin molecules, and their molecular weight is larger than that of a single cyclodextrin molecule, which is not conducive to transdermal absorption. Therefore, the exploration of cyclodextrin-artemether inclusion complexes with relatively small molecular weight is not only easy to be absorbed through the skin, but also can stably release drugs. These characteristics are extremely important for the development of shampoos. It has both antibacterial and anti-inflammatory effects in the treatment of seborrheic dermatitis of the scalp and tinea versicolor on the trunk and other Pityrosporum infections.

[0003] In addition to its anti-malarial effect, artemether also has anti-inflammatory effects. Many experimental studies at home and abroad have shown that artemisinin and its derivatives can inhibit the release of downstream pro-inflammatory factors by blocking several upstream signal transduction pathways of NF-κB. [1] , including IL-2, IL-6, IFNγ and other cytokines [2-5] It has been clinically used to treat immune-related diseases such as rheumatoid arthritis, systemic lupus erythematosus, and rosacea.

[0004] The applicant has long been committed to the research of artemisinin and its derivatives. In recent years, our team has found that (1) artemisinin can inhibit the activity of Demodex folliculorum. [6] , and topical artemether emulsion is effective in treating rosacea with a low recurrence rate [7] , (2) Artemether has a significant inhibitory effect on Propionibacterium acnes [8] It is also effective in treating acne [9] (3) Recent studies have found that seborrheic dermatitis is associated with infection with Pityrosporum fumigatus, Propionibacterium acnes, and Demodex folliculorum.

[10] When we were testing artemether shampoo, we found that artemether had low water solubility and lipid solubility, and it was difficult to prepare shampoo with effective concentration. Therefore, we conducted research on artemether-cyclodextrin inclusion complex to improve its solubility for the development of anti-inflammatory shampoo.

[0005] Cyclodextrin is a type of glucose unit, each unit is connected by glycosidic bonds to form a "cylindrical" structure. The hydroxyl group of the molecule is located outside the barrel structure and is hydrophilic; while the hydrogen atom is located in the cavity of the cylindrical structure, which has the hydrophobic property of the cavity and can accommodate hydrophobic compounds of suitable molecular weight to form an inclusion complex. This inclusion complex improves the solubility in aqueous solution. Cyclodextrins containing 6, 7, and 8 glucose units are called α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin, respectively. It is generally believed that the cavity of β-cyclodextrin can accommodate molecules less than 400 dol, and the molecular weight of artemether is 298.37, which can be used to make the inclusion complex. Summary of the invention

[0006] In order to overcome the shortcoming of low water solubility of artemether in shampoo, the present invention firstly needs to provide a method for preparing a water-soluble artemether-cyclodextrin inclusion complex.

[0007] Another object of the present invention is to provide an artemether-cyclodextrin inclusion complex that can improve the water solubility and compatibility of artemether and is used for preparing shampoo emulsions.

[0008] The water-soluble artemether-cyclodextrin inclusion compound provided by the present invention consists of artemether, cyclodextrin and a complex.

[0009] The water-soluble theophylline-cyclodextrin inclusion compound provided by the present invention comprises the following components by mass percentage: 15-20% of artemether, 60-70% of cyclodextrin and 15-20% of ethylenediaminetetraacetic acid.

[0010] Preferably, in the water-soluble artemether-cyclodextrin inclusion compound composition, the molar ratio of artemether to cyclodextrin is 1:1.

[0011] The cyclodextrin is preferably β-cyclodextrin.

[0012] The complexing agent can be selected from one or more of ethylenediaminetetraacetic acid (EDTA), sodium polyacrylate (PAAS), and polyvinylpyrrolidone (PVP), preferably ethylenediaminetetraacetic acid (EDTA.

[0013] The particle diameter of artemether-cyclodextrin inclusion complex is 30-180nm.

[0014] The method for preparing artemether-cyclodextrin inclusion complex provided by the present invention comprises the following steps.

[0015] (1) Add artemether to anhydrous ethanol, mix well and fully dissolve to prepare an artemether ethanol solution.

[0016] (2) Add cyclodextrin and a complexing agent into deionized water and mix well to obtain a cyclodextrin complex aqueous solution; then add artemether ethanol solution while stirring and stir well to obtain a mixed solution.

[0017] (3) The mixed solution is evaporated to remove ethanol to obtain an artemether-cyclodextrin inclusion complex, which is then freeze-dried to obtain a water-soluble artemether-cyclodextrin inclusion complex.

[0018] In the artemether ethanol solution, the concentration of artemether is 25-75mM, and preferably the concentration of artemether is 50mM.

[0019] The cyclodextrin aqueous solution has a cyclodextrin concentration of 25-200 mM, preferably 50 mM.

[0020] In the cyclodextrin aqueous solution, the concentration of the complex is 20-50 mM, preferably 50 mM.

[0021] The volume ratio of the cyclodextrin aqueous solution to the artemether ethanol solution is 1:1.

[0022] The stirring temperature is 35-55 0 C, the stirring time is 12-48 hours, and the stirring rate is 200-800r / min. Preferably, the mixing temperature is 37 0 C, the stirring and mixing time is 24 hours, and the stirring and mixing rate is 700r / min.

[0023] Compared with the prior art, the advantages of the present invention are as follows: the present invention has a water-soluble artemether-cyclodextrin inclusion complex, which has improved solubility in aqueous solution compared with artemether and has good stability.

[0024] The water-soluble artemether and cyclodextrin inclusion compound of the present invention can be compounded with various shampoo raw materials and shows good compatibility.

[0025] The present invention utilizes artemether-cyclodextrin inclusion compound technology and provides a method for preparing the artemether-cyclodextrin inclusion compound. The method has a simple preparation process and a short time, and is conducive to industrial production.

[0026] Antibacterial effect of artemether-cyclodextrin inclusion complex against standard strains of Pityrosporum fumigatus.

[0027] The experimental drug artemether was purchased from Huawu Pharmaceutical; ketoconazole was purchased from Shanghai MacLean Biochemical Technology Co., Ltd.

[0028] The experimental strain Malassezia furfur standard strain (Malassezia furfur), strain number: BNCC 337308, was purchased from Beijing Beina Chuanglian Biotechnology Research Institute (China General Microbial Culture Conservation Center).

[0029] Experimental reagents: malt extract powder was purchased from Beijing Aoboxing Biotechnology Co., Ltd.; ox bile powder, glycerol monooleate, Tween 40, agar, maltose, yeast extract, and glucose were purchased from Shanghai MacLean Biochemical Technology Co., Ltd. Dimethyl sulfoxide was purchased from Mingke Experimental Equipment Business Department, Mingguang City, Anhui Province.

[0030] Experimental instruments: UTP-313 electronic balance (Shanghai Huachao Electric Co., Ltd.); DNP-9082 electric constant temperature incubator (Shanghai Jinghong Experimental Equipment Co., Ltd.); BSC-1100ⅡB2-X biological safety cabinet (Jinan Xinbeisi Biotechnology Co., Ltd.); XFS-280CB portable pressure steam sterilizer (Zhejiang Xinfeng Medical Equipment Co., Ltd.); ZC-MCF McFarland turbidimeter (Beijing Zhecheng Technology Co., Ltd.).

[0031] The formula of Malassezia solid culture medium is malt extract powder 40g, ox bile powder 20g, glycerol monooleate 2.5g, Tween 40 10g, agar 15g, and distilled water 1.0L; Malassezia liquid culture medium: maltose 20g, yeast extract 2g, glucose 20g, olive oil 20g, and distilled water 1.0L.

[0032] Preparation of culture medium and activation of strains Weigh each component according to the above proportions, sterilize at 121℃ for 15min after it is fully thawed, and prepare Malassezia solid culture medium. Activation of standard strains of Malassezia furfur Dissolve the bacterial powder in an appropriate amount of sterile saline, evenly spread it on the solid culture medium, and place it in a 30℃ incubator.

[0033] Preparation of bacterial suspension: The standard strain of Malassezia furfur was inoculated on solid culture medium and subcultured once every 72 hours for two times to prepare bacterial suspension. The suspension was adjusted to 0.5 McFarland units using McFarland turbidimeter and then diluted 10 times with liquid culture medium. At this time, the bacterial content was (1.0-2.0) x10 6 CFU / mL.

[0034] To prepare the drug solution, artemether was diluted to 10000 μg / mL with DMSO, ketoconazole was diluted to 250 μg / mL with DMSO, and then diluted ten-fold with pure water, and then diluted in multiple ratios.

[0035] Determination of antibacterial activity: Inoculate 0.1 mL of the diluted bacterial suspension on the surface of the Malassezia solid culture medium plate and spread it evenly, and punch holes with a 6-mm diameter sterile puncher; dilute artemether and ketoconazole into different concentrations of drug solutions and add them into different holes until they are full, and then culture them in a 30°C constant temperature incubator for 48 hours.

[0036] Interpretation of solid culture results: Use a digital display vernier caliper to measure the diameter of the inhibition zone, and calculate the average and standard deviation of the diameter of the inhibition zone of each drug solution at different concentrations. In-house quality control: The positive control is inoculated with bacterial suspension, without drug solution; the negative control is not inoculated with bacterial suspension.

[0037] Preparation and inoculation of microplates Take a 96-well microplate and add 100 μL of culture medium to the 2nd to 11th wells, add 200 μL of drug solution to the 1st well, and according to the doubling dilution method, aspirate 100 μL of drug solution from the 1st well of each row and add it to the corresponding 2nd well. After mixing evenly, aspirate 100 μL of drug solution from the 2nd well and add it to the corresponding 3rd well. And so on. After mixing evenly in the 10th well, discard the aspirated 100 μL of drug solution. Add 100 μL of prepared bacterial solution to each well from the 1st to the 11th column. The 11th well is a positive control with liquid culture medium and bacterial suspension, and the 12th well is a negative control with only liquid culture medium. Finally, add 100 μL of Malassezia suspension to each well. The first row is the artemether group and the second row is the ketoconazole group. The concentrations of artemether were 10000, 5000, 2500, 1250, 625, 312.5, 156, 78, 39, and 19.5 μg / mL, and the concentrations of ketoconazole were 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, and 1.95 μg / mL. The final concentration of DMSO was <1%. After control experiments, this concentration could not inhibit the growth of Malassezia. The above experimental plates were placed in a 30°C incubator for 48 hours.

[0038] Observe the colony growth 48 hours after the micro-drug sensitivity plate results are read. The positive control wells with good growth are taken as the standard, and the lowest drug concentration well position with no visible bacterial growth is taken as the MIC of the test. The quality control wells do not contain drug solution, and 100μL of bacterial suspension is added as the positive control well. 100uL of drug solution without bacterial suspension is used as the negative control well.

[0039] The effects of artemether and ketoconazole on Malassezia were expressed as inhibition zone diameter and MIC. The data were statistically analyzed using EXCEL and the quantitative data were expressed as X+S.

[0040] Agar plate diffusion method (punch method) experimental results Figure 1 Some photos showing the antibacterial effects of different concentrations of artemether and ketoconazole on Malassezia using the agar plate diffusion method (punch method). The antibacterial diameters of the above drugs were measured successively, and the experiment was repeated 3 times. The results are shown in Table 1.

[0041] Table 1 The inhibitory diameter of three artemisinin drugs against Malassezia furfur (X±S) mm

[0042] Micro-liquid dilution test results The drug sensitivity of artemether in 96-well plate is shown in Table 2. The surface of the liquid in the well is clear and sterile growth is indicated by "-", and bacterial growth is indicated by "+". No drug solution was added to the positive control well, and no bacterial solution was added to the negative control well. The MIC of Artemisia annua extract is 10000μg (10mg) / mL, the MIC of artesunate is 625μg / mL, the MIC of artemether is 625μg / mL, and the MIC of ketoconazole in the control group is 31.25μg / mL. See Table 2 for details. Table 2 Minimum inhibitory concentration of three artemisia drugs against Malassezia furfur Table 2 Minimum inhibitory concentrations of three artemisia annua drugs against Malassezia furfur

[0043] Conclusion: This study confirmed that artemether has different degrees of inhibitory effect on Malassezia in vitro through agar plate diffusion method and micro-liquid dilution method. Its antibacterial effect is positively correlated with drug concentration. The MIC of artemether is 625μg / mL, which is significantly lower than that reported in the literature for Microsporum gypseum, Trichophyton roseum, Trichophyton rubrum, etc. It provides a basis for the use of artemether shampoo in the treatment of scalp seborrheic dermatitis. Artemether has antifungal and anti-inflammatory effects, making it superior to other antifungal drugs.

[0044] References [1] Cheng Lu. Comparative study on the antifungal susceptibility of the same Malassezia species isolated from different diseases. Graduate thesis of Kunming University, published in January-February 2018.

[0045] [2] Luo Wanting, Li Haitao, Yang Rongya. Research progress on antifungal effects of artemisinin and its derivatives. Journal of Practical Dermatology, 2021, 14(1):36-38. [3] Liu Di, Guo Qingquan, Zhao Haishan, et al. Study on the anti-Malassezia activity, cytotoxicity and antibacterial mechanism of eugenol. Journal of Guangdong University of Technology, 2021, 38(02):88-93. [4] Li Jun, Guo Hongyan, Chen Haiyan. Study on the in vitro antibacterial activity of Sophora alopecuroides extract against Malassezia. Light Industry Science and Technology, 2023, 39(1): 10-14. [5] Fu Rong, Li Yuanwen, Zhou Zhiqiang, et al. In vitro drug sensitivity test of five traditional Chinese medicines including Radix Glehniae and their different combinations against Malassezia furfur. Global Chinese Medicine, 2015, 8(10): 1164-1167. [6] Wang Jie, Tang Chunping, Jiang Tao. Study on the antifungal effect of the effective fraction of Dryopteris odorifera against Malassezia furfur in vitro. Chinese Traditional Patent Medicine. 2015, 37(3): 642-645. [7] Marinas IC, Oprea E, Chifiriuc MC, Badea IA, Buleandra M, Lazar V. Chemical Composition and Antipathogenic Activity of Artemisia annua EssentialOil from Romania. Chem Biodivers. 2015 Oct;12(10):1554-64. doi: 10.1002 / cbdv.201400340. [8] Chen Deyu, Zheng Jiuan, Huang Shufang. Experimental study on the antifungal effect of Artemisia annua oil liniment and observation of its efficacy [J]. Chinese Journal of Dermatology and Venereology, 1990, 01: 18-19. [9] Liu Bifeng, Ye Qili, Zhao Yi. Experimental study on the effect of artesunate sodium on microorganisms[J]. Chinese Pharmacological Bulletin, 1986, 2(3):26-29.

[10] Jin Huiling, Zhang Ruzhi, Gao Yuxiang, et al. Experimental study on antifungal and antibacterial effects of artesunate [J]. Chinese Journal of Microecology, 2003, 15(1): 26-27.

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Claims

1. A method for preparing a water-soluble artemether-cyclodextrin inclusion complex, wherein the artemether-cyclodextrin inclusion complex powder contains 17.29% artemether, 65.78% β-cyclodextrin, and 16.93% ethylenediaminetetraacetic acid in terms of mass percentage. The method for preparing the water-soluble artemether-cyclodextrin inclusion complex is as follows: weigh 0.746 g artemether (molecular weight 298.37), add anhydrous ethanol to 50 mL, and mix to obtain an artemether ethanol solution with a concentration of 50 mM. Weigh 2.837 g of β-cyclodextrin (molecular weight 1134.98) and 0.73 g of EDTA (molecular weight 292.24), put them into the same test tube, add double distilled water to 50 mL, mix and dissolve them thoroughly to obtain a 50 mM aqueous solution of β-cyclodextrin (containing EDTA concentration of 50 mM), take 5 mL of the above artemether ethanol solution and 5 mL of β-cyclodextrin EDTA aqueous solution, mix the two liquids, and heat at 37 0 C ambient temperature, 700r / min centrifugation for 24 hours; then evaporate the solution to remove ethanol to obtain an aqueous solution of artemether-cyclodextrin inclusion complex, and place in a refrigerator at -18 0 C and freeze until the water is fully volatilized to obtain dry powder, which is artemether-cyclodextrin inclusion complex. The concentration of artemether in shampoo products is 1-50g / L.

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