A cedrol nanosuspension and its preparation and application

Abstract

This invention discloses a cedrol nanosuspension, its preparation, and its application. The cedrol nanosuspension comprises cedrol, a stabilizer, a solvent, and an antisolvent. This invention uses PEG 400 as the solvent, avoiding the use of traditional organic solvents, reducing skin irritation, and providing an environmentally friendly method for preparing the nanosuspension. This invention selects SDS and HPMC as a composite stabilizer; the two exert a complementary stabilizing mechanism in the nanosuspension, further increasing the system's stability. The cedrol nanosuspension described in this invention can promote AGA hair regeneration, providing a new approach for developing novel topical hair growth agents with high efficacy and low side effects.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical formulation technology, specifically relating to a cedrol nanosuspension and its preparation and application. Background Technology

[0002] Androgenetic alopecia (AGA), also known as seborrheic alopecia, is the most common type of hair loss. It is characterized by abnormal hair growth cycles, specifically including gradual shrinkage of hair follicles, shortened growth cycles, and prolonged resting phases. In the pathogenesis of AGA, androgens are considered a core factor driving disease progression. Testosterone, the main androgen in the body, is converted into the more active 5α-dihydrotestosterone (DHT) in hair follicles by 5α-reductase. DHT binds to androgen receptors in the hair follicles, leading to gradual follicle shrinkage, shortened hair growth cycles, and ultimately hair loss. Minoxidil, a first-line clinical drug, is widely recommended for promoting hair growth and is usually administered topically. Oral administration of minoxidil may cause tachycardia, pericarditis, nausea, hirsutism, orthostatic blood pressure fluctuations, and edema. Topical application of minoxidil, in addition to reduced absorption, can cause scalp irritation, dandruff, scalp itching, and allergies.

[0003] Cedrol (CE), also known as cedrol or cedrol alcohol, is a natural sesquiterpene alcohol compound that can be extracted from various plants in the Cupressaceae, Taxaceae, and Pinaceae families. It possesses pharmacological effects such as anti-anxiety, anti-inflammatory and analgesic properties, effects on the cardiovascular system, anti-tumor activity, and antibacterial activity. However, cedrol is poorly soluble in water, which limits its clinical development and application.

[0004] Therefore, there is a need to develop cedrol-related formulations suitable for clinical use in order to improve the therapeutic effect of cedrol drugs. Summary of the Invention

[0005] The present invention aims to at least partially solve one of the aforementioned technical problems or at least provide a useful commercial solution. To this end, the present invention provides a cedrol nanosuspension, its preparation, and its application.

[0006] In a first aspect, the present invention provides a cedrol nanosuspension. According to embodiments of the present invention, the cedrol nanosuspension comprises: cedrol, a stabilizer, a solvent, and an antisolvent. The cedrol nanosuspension of the present invention has a scientifically sound formulation and proportioning, a simple preparation process, controllable quality, and is convenient to carry and use. Its suitable consistency facilitates drug release, promotes formulation and molding, and ensures the safety, efficacy, and quality control of the cedrol nanosuspension in clinical application, thereby improving the therapeutic effect of cedrol as a topical medication.

[0007] According to an embodiment of the present invention, the stabilizer of the cedrol nano-suspension of the present invention is selected from at least one of the following: sodium dodecyl sulfate (SDS), polyvinylpyrrolidone K30 (PVP K30), poloxamer 407 (P407), hydroxypropyl methylcellulose (HPMC), Tween-80 (TW-80), and sodium carboxymethyl cellulose (CMC-Na).

[0008] According to embodiments of the present invention, the stabilizer of the cedrol nano-suspension of the present invention is preferably at least one of sodium dodecyl sulfate, poloxamer 407, Tween-80, and hydroxypropyl methylcellulose.

[0009] According to embodiments of the present invention, it is further preferred that the stabilizer of the cedrol nano-suspension of the present invention is at least one of sodium dodecyl sulfate and hydroxypropyl methylcellulose; it is further preferred that the stabilizer of the cedrol nano-suspension of the present invention is a combination of sodium dodecyl sulfate and hydroxypropyl methylcellulose; it is further preferred that the weight ratio (g / g) of the combination of sodium dodecyl sulfate and hydroxypropyl methylcellulose is (0.5~2.0):1; it is further preferred that the weight ratio of the combination of sodium dodecyl sulfate and hydroxypropyl methylcellulose is (1.0~1.5):1; it is further preferred that the weight ratio of the combination of sodium dodecyl sulfate and hydroxypropyl methylcellulose is 1.31:1.

[0010] According to an embodiment of the present invention, the solvent of the cedrol nano-suspension of the present invention is selected from polyethylene glycol 400 (PEG400).

[0011] According to an embodiment of the present invention, the antisolvent for the cedrol nano-suspension of the present invention is selected from water.

[0012] According to embodiments of the present invention, the water described herein includes, but is not limited to, purified water, water for injection, etc.

[0013] According to an embodiment of the present invention, in the cedrol nano-suspension of the present invention, the weight ratio of solvent to antisolvent is 1:(10~15), preferably 1:12.

[0014] According to an embodiment of the present invention, the formulation of the cedrol nano-suspension of the present invention comprises, per 100 parts by weight: 0.5 to 1.0 parts by weight of cedrol, 0.3 parts by weight of stabilizer, 6.17 to 9.02 parts by weight of solvent, and 89.73 to 93.00 parts by weight of antisolvent.

[0015] According to an embodiment of the present invention, the formulation of the cedrol nano-suspension of the present invention comprises, per 100 parts by weight: 0.5-1.0 parts by weight of cedrol, 0.15-0.18 parts by weight of SDS, 0.12-0.15 parts by weight of HPMC, 6.17-9.02 parts by weight of PEG400, and 89.73-93.00 parts by weight of water.

[0016] According to a specific embodiment of the present invention, the cedrol nano-suspension formulation of the present invention comprises, per 100 parts by weight: 0.75 parts by weight of cedrol, 0.17 parts by weight of SDS, 0.13 parts by weight of HPMC, 7.60 parts by weight of PEG400, and 91.35 parts by weight of water.

[0017] According to a specific embodiment of the present invention, the cedrol nano-suspension formulation of the present invention comprises, per 100 parts by weight: 0.75 parts by weight of cedrol, 0.15 parts by weight of SDS, 0.15 parts by weight of HPMC, 9.00 parts by weight of PEG400, and 89.95 parts by weight of water.

[0018] According to a specific embodiment of the present invention, the cedrol nano-suspension formulation of the present invention comprises, per 100 parts by weight: 0.75 parts by weight of cedrol, 0.18 parts by weight of SDS, 0.12 parts by weight of HPMC, 6.18 parts by weight of PEG400, and 92.77 parts by weight of water.

[0019] According to a specific embodiment of the present invention, the cedrol nano-suspension formulation of the present invention comprises, per 100 parts by weight: 0.50 parts by weight of cedrol, 0.17 parts by weight of SDS, 0.13 parts by weight of HPMC, 7.63 parts by weight of PEG400, and 91.57 parts by weight of water.

[0020] According to a specific embodiment of the present invention, the cedrol nano-suspension formulation of the present invention comprises, per 100 parts by weight: 0.50 parts by weight of cedrol, 0.15 parts by weight of SDS, 0.15 parts by weight of HPMC, 9.02 parts by weight of PEG400, and 90.18 parts by weight of water.

[0021] According to a specific embodiment of the present invention, the cedrol nano-suspension formulation of the present invention comprises, per 100 parts by weight: 0.50 parts by weight of cedrol, 0.18 parts by weight of SDS, 0.12 parts by weight of HPMC, 6.20 parts by weight of PEG400, and 93.00 parts by weight of water.

[0022] According to a specific embodiment of the present invention, the cedrol nano-suspension formulation of the present invention comprises, per 100 parts by weight: 1.0 part by weight of cedrol, 0.17 parts by weight of SDS, 0.13 parts by weight of HPMC, 7.59 parts by weight of PEG400, and 91.11 parts by weight of water.

[0023] According to a specific embodiment of the present invention, the cedrol nano-suspension formulation of the present invention comprises, per 100 parts by weight: 1.0 part by weight of cedrol, 0.15 parts by weight of SDS, 0.15 parts by weight of HPMC, 8.97 parts by weight of PEG400, and 89.73 parts by weight of water.

[0024] According to a specific embodiment of the present invention, the cedrol nano-suspension formulation of the present invention comprises, per 100 parts by weight: 1.0 part by weight of cedrol, 0.18 parts by weight of SDS, 0.12 parts by weight of HPMC, 6.17 parts by weight of PEG400, and 92.53 parts by weight of water.

[0025] The weight units mentioned in this invention include, but are not limited to, micrograms, milligrams, grams, kilograms, and tons.

[0026] In a second aspect, the present invention provides a method for preparing cedrol nanosuspension.

[0027] According to an embodiment of the present invention, the preparation method of the cedrol nano-suspension of the present invention comprises the following steps: Formulation: Per 100g contains: 0.5~1.0g cedrol, 0.15~0.18g SDS, 0.12~0.15g HPMC, 6.17~9.02g PEG400, and 89.73~93.00g water; Step (1): Accurately weigh cedrol and place it in a beaker equipped with a magnetic stir bar. Add PEG400 and stir continuously in a constant temperature water bath at 90±2℃ until cedrol is completely dissolved to form a clear, homogeneous, and transparent drug solution, which is used as the drug phase. After cooling at room temperature, it is ready for use. Step (2): Add SDS and HPMC to purified water in sequence, and stir at 300~500 r / min for 10~20 min at room temperature until SDS and HPMC are completely dissolved to form a homogeneous, colorless and transparent aqueous solution, which is used as the aqueous phase and placed in an ice-water bath for later use. Step (3): The drug phase in step (1) is added dropwise to the aqueous phase in step (2) at a rate of 18 mL / min to 22 mL / min using a peristaltic pump. During the dropwise addition, a high-speed shear dispersion emulsifier is used to continuously shear and emulsify at a speed of 9000 r / min to 11000 r / min. After the drug phase is added, the shearing is continued at the same speed for 4 to 6 minutes to obtain the cedrol nano suspension of the present invention.

[0028] According to a specific embodiment of the present invention, the preferred method for preparing the cedrol nano-suspension comprises the following steps: Formulation: Per 100g contains: 0.75g cedrol, 0.17g SDS, 0.13v HPMC, 7.60g PEG400, and 91.35g water.

[0029] Step (1): Accurately weigh 0.75g of cedrol and place it in a beaker equipped with a magnetic stir bar. Add 7.60g of PEG400 and place it in a constant temperature water bath at 90±2℃. Continue stirring until the cedrol is completely dissolved to form a clear, homogeneous, and transparent drug solution, which is used as the drug phase. Allow it to cool at room temperature for later use. Step (2): Add 0.17g SDS and 0.13g HPMC to 91.35g of purified water in sequence. Stir at 300~500 r / min for 10~20 min at room temperature until SDS and HPMC are completely dissolved to form a homogeneous, colorless and transparent aqueous solution. Use this as the aqueous phase and place it in an ice-water bath for later use. Step (3): The drug phase in step (1) is added dropwise to the aqueous phase in step (2) at a rate of 20 mL / min using a peristaltic pump. During the dropwise addition, a high-speed shear dispersion emulsifier is used to continuously shear and emulsify at a speed of 10,000 r / min. After the drug phase is completely added, the shearing is continued at the same speed for 5 min to obtain the cedrol nano suspension of the present invention.

[0030] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention. All reagents and raw materials used in the present invention are commercially available.

[0031] According to a third aspect of the present invention, a method for determining the cedrol content in a cedrol nanosuspension is provided. This method for determining the cedrol content in a cedrol nanosuspension is an independently developed method.

[0032] A method for determining the cedrol content in cedrol nanosuspension (CE-NS), characterized in that the method utilizes high-performance liquid chromatography (HPLC) combined with an ultraviolet detector, wherein... (1) The chromatographic conditions are as follows: the chromatographic column is a C8 alkyl-bonded silica reversed-phase column; the mobile phase consists of acetonitrile-0.1% phosphoric acid aqueous solution; the detection wavelength is 190 nm ± 2 nm; the column temperature is 25~35℃; the flow rate is 0.8~1.2 mL / min; and the injection volume is 5~20 μL. Optionally, the chromatographic conditions are as follows: column: Fortis XI C8 (250 × 4.6 mm, 5 μm); mobile phase: acetonitrile-0.1% phosphoric acid aqueous solution (the volume ratio of acetonitrile to 0.1% phosphoric acid aqueous solution is 85:15); detection wavelength: 190 nm; column temperature: 30℃; flow rate: 1.0 mL / min; injection volume: 10 μL.

[0033] (2) Preparation of solutions: Prepare cedrol reference solution, CE-NS test solution and negative sample solution respectively; Further, in step (2), the preparation of the cedrol reference solution is as follows: accurately weigh 10 mg of cedrol reference standard, place it in a 50 mL volumetric flask, add a certain amount of acetonitrile to dissolve it by sonication, and dilute to the mark to obtain a 200 μg / mL cedrol reference solution, which is then sealed and stored at 4 ℃ for later use; the preparation of the test solution is as follows: accurately pipette 1 mL of CE-NS suspension into a 50 mL volumetric flask, add acetonitrile to dissolve it by sonication, and dilute to the mark to obtain a CE-NS test solution; the preparation of the negative sample solution is as follows: take a negative sample that does not contain cedrol, the negative sample being composed of SDS, HPMC, PEG400 and water, and prepare it according to the same method as the test solution to obtain the negative sample solution; (3) Determination: Inject the reference solution, test solution and negative sample solution from step (2) above into the high performance liquid chromatograph, and perform the determination according to the chromatographic conditions of step (1) above. Record the corresponding chromatograms. Calculate the content of cedrol in the test sample by peak area according to the external standard method. There should be no interfering peak at the retention time of the cedrol reference in the chromatogram of the negative sample solution.

[0034] The analytical detection method described in this invention demonstrates good specificity through specificity studies. Linearity studies show good linearity of cedrol within the range of 5–200 μg / mL. Precision studies indicate good instrument precision. Stability studies show good stability of the CE-NS test solution within 24 h. Repeatability studies show good repeatability. Recovery studies show high accuracy. Specifically, the results are as follows: Specificity assessment: According to the chromatographic conditions of step (1) above, cedrol reference solution, CE-NS test solution and negative control solution were respectively subjected to HPLC detection. The results showed that the chromatographic peak of cedrol was not affected by other components, and the method has good specificity.

[0035] Linearity assessment: Accurately pipette the cedrol reference solution and dilute it with acetonitrile to prepare a series of cedrol reference solutions with mass concentrations of 5, 10, 25, 50, 100, and 200 μg / mL. Perform the chromatographic determination under the conditions described in step (1) above, and record the peak areas. Plot the mass concentration as the abscissa (X) and the peak area as the ordinate (Y), and perform linear regression analysis. The linear regression equation is Y = 551.71X + 598.12, R0. 2 =0.9998, indicating that cedrol showed good linearity in the range of 5–200 μg / mL.

[0036] Precision test: A 100 μg / mL cedrol reference solution was injected and measured 6 times consecutively under the chromatographic conditions described in (1) above. The results showed that the peak area of ​​cedrol was 0.62%, indicating that the instrument had good precision.

[0037] Stability study: The CE-NS test solution was tested at 0, 2, 4, 8, 12 and 24 h after preparation, respectively, under the chromatographic conditions of step (1) above. The RSD of the peak area of ​​cedrol was calculated to be 1.61%, which shows that the CE-NS test solution has good stability within 24 h.

[0038] Repeatability test: Take CE-NS suspension and prepare 6 test solutions according to the above method (3). Determine the RSD of cedrol mass fraction according to the above step (1). The results show that the method has good repeatability.

[0039] Recovery rate study: Nine 0.5 mL aliquots of CE-NS suspension were placed in 25 mL volumetric flasks and divided into three groups: low, medium, and high. Cedrol reference standard was added at levels of 80%, 100%, and 120%, respectively. The cedrol content was determined and the recovery rate was calculated under the chromatographic conditions described in step (1) above. The results showed that the average recovery rate of CE-NS was 100.41%, and the RSD was 0.86%, indicating that the method has high accuracy.

[0040] In a fourth aspect, the present invention provides the use of a cedrol nanosuspension that promotes hair regeneration in androgenetic alopecia. According to embodiments of the present invention, the cedrol nanosuspension is used to prepare a medicament for promoting hair regeneration in androgenetic alopecia. The cedrol nanosuspension prepared according to embodiments of the present invention, when applied to the skin for 48 hours, exhibits no significant skin irritation or allergic reactions.

[0041] The positive and progressive effects of this invention are as follows: (1) This invention uses a solvent-antisolvent precipitation method to prepare CE-NS, which improves upon the traditional precipitation method in terms of formulation design. The traditional precipitation method involves dissolving the drug in an organic solvent and simultaneously dissolving the stabilizer in an antisolvent (usually water), forming drug particles through crystallization. However, this method easily results in solvent residue in the final formulation and poor physical stability, leading to sedimentation and aggregation, resulting in low drug loading. This invention uses PEG 400 as a solvent, which avoids the use of traditional organic solvents, reduces skin irritation, and provides an environmentally friendly method for preparing nano-suspensions.

[0042] (2) The inventors of this invention have for the first time prepared cedrol nano-suspension. In the preparation process of NS, the type and amount of stabilizer are key factors in maintaining the stability of drug nanoparticles and directly affect the physical stability of NS. The single-factor experimental results of the embodiments of this invention show that although SDS or HPMC can be used alone to prepare suspensions, the particle size and PDI are large, and the stability and redispersibility of nano-suspension are poor when SDS is used alone. Therefore, this invention selects SDS and HPMC as composite stabilizers. The two play a complementary stabilizing mechanism in nano-suspension, further increasing the stability of the system. In this study, response surface methodology was used for system optimization in the preparation process to ensure that the final formulation process is scientific and reproducible.

[0043] (3) The in vitro transdermal test results of the embodiments of the present invention showed that no active drug was detected in the receiving solution for either the free drug or CE-NS within 24 hours. However, the skin retention of CE-NS was significantly higher than that of the free drug, which was 4.1 times that of the free drug. This indicates that the cedrol nano-suspension with a particle size in the range of 420~470 nm prepared by the present invention can effectively reduce the risk of drug entering the systemic circulation, reduce side effects, and increase the accumulation of drug in the skin, thereby enhancing the local therapeutic effect. The results show the superiority of the nano-suspension delivery system in improving drug retention in the skin. From macroscopic hair coverage, length and weight to microscopic histological observation, the present invention has once again demonstrated that CE-NS can effectively promote the accumulation of cedrol in the hair follicle area through the transdermal delivery system, thereby accelerating the hair follicle growth cycle and promoting hair regeneration. The transdermal drug delivery scheme of the cedrol nano-suspension described in the present invention can promote AGA hair regeneration and provides a new idea for developing novel topical hair growth preparations with high efficiency and low side effects. Attached Figure Description

[0044] Figure 1 The HPLC chromatograms are of the cedrol reference standard (A), CE-NS sample (B), and blank sample (C) solutions described in Example 1 of this invention. Figure 2 This is a particle size distribution diagram of the cedrol nano-suspension described in Example 1 of the present invention; Figure 3 The skin retention amount of CE-NS in Example 11 of the present invention; Figure 4 The ATR-FTIR results of pigskin treated with different samples as described in Example 11 of this invention; Figure 5 These are SEM scans of pig skin treated with different formulations as described in Example 11 of this invention (A, B: PBS solution; C, D: CE-NS). Figure 6 This refers to the changes in body weight of mice in each group as described in Example 12 of the present invention; Figure 7 This describes the hair growth and skin color of mice in different groups at different time periods as described in Example 12 of the present invention; Figure 8 The hair coverage rate of each group of mice described in Example 12 of this invention; Figure 9 This refers to the examination of skin tissues in each group of mice as described in Example 12 of the present invention; Figure 10 The immunohistochemical staining results of AR, Ki67, and β-catenin in the skin tissues of each group of mice described in Example 12 of this invention; Figure 11This is the HPLC detection chromatogram described in Comparative Example 1 of the present invention; Figure 12 This is the HPLC detection chromatogram described in Comparative Example 2 of the present invention; Figure 13 This is the ultraviolet-visible absorption spectrum described in Comparative Example 2 of the present invention; Figure 14 This is the HPLC detection chromatogram described in Comparative Example 3 of the present invention; Figure 15 This is the HPLC detection chromatogram described in Comparative Example 4 of the present invention; Figure 16 This is the HPLC detection chromatogram described in Comparative Example 5 of the present invention. Detailed Implementation

[0045] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0046] Instruments: ME104E electronic balance (Mettler-Toledo Instruments Ltd.); DF-101S thermostatic magnetic stirrer (Zhengzhou Changcheng Science & Industry Co., Ltd.); YZ1515X peristaltic pump (Baoding Chuangrui Pump Industry Co., Ltd.); FA25D digital display high-shear dispersion emulsifier (Shanghai Fluke Technology Development Co., Ltd.); ST2100 pH meter (Ohaus Instruments Ltd.); ZS90 nanoparticle size potentiometer (Malvin Panaco); LC-20A high performance liquid chromatograph (Shimadzu Corporation, Japan).

[0047] Reagents and test drugs: Cedrol raw material (batch number 20240315, mass fraction 97.05%, Jiangxi Baolin Natural Flavors Co., Ltd.); Cedrol reference substance (batch number D2407033, mass fraction > 98%, Shanghai Aladdin Biochemical Technology Co., Ltd.); PEG400 (batch number 20220928, Sinopharm Chemical Reagent Co., Ltd.); Sodium dodecyl sulfate (SDS, batch number F310C231002, Hubei Gedian Humanwell Pharmaceutical Excipients Co., Ltd.); Polyvinylpyrrolidone K30 (PVP K30, batch number C15050213, Shanghai Macklin Biochemical Co., Ltd.); Poloxamer 407 (P407, batch number H11J9C52602, Shanghai Yuanye Bio-Technology Co., Ltd.); Tween 80 (batch number D0277HB35, Guangdong Run Chemical Co., Ltd.); Hydroxypropyl methylcellulose (HPMC, batch number 2405018, Shandong Yiteng Materials Co., Ltd.); Sodium carboxymethyl cellulose (CMC-Na, batch number 30036328, Sinopharm Chemical Reagent Co., Ltd.), and other reagents are of analytical grade.

[0048] Animals: 60 SPF-grade male C57BL / 6 mice, 7 - 8 weeks old, weighing 20 - 22 g, purchased from Hubei Bente Biotechnology Co., Ltd., experimental animal license number: SCXK(E)2021 - 0027. The animals were housed in the Experimental Animal Center of Hubei University of Chinese Medicine. After 1 week of adaptive feeding in an environment with suitable temperature, humidity and light, the mice were subjected to model establishment. During the experiment, the mice were allowed to eat, drink and move freely.

[0049] Example 1 Prescription screening, preparation, analysis and determination of cedrol nanosuspension (CE-NS) 1.1 Preparation of CE-NS CE-NS was prepared by the solvent-antisolvent precipitation method. Weigh the prescribed amount of CE and dissolve it in the PEG400 solution, and then dissolve it by ultrasonic treatment in a boiling water bath to obtain the drug solvent phase; dissolve SDS and HPMC in pure water and fully swell them to obtain the antisolvent phase; add the CE solution phase to the antisolvent phase at a certain speed through a peristaltic pump, and continuously stir during the addition process. After the solution is completely added, continue to stir to obtain CE-NS.

[0050] 1.2 Determination of the particle size, polydispersity index (PDI) and Zeta potential of CE-NS Take 1 mL of the CE-NS sample and transfer it to a cuvette. Use a Malvern nano laser particle size analyzer to measure the particle size, the PDI value reflecting the particle size distribution and the Zeta potential. Repeat the measurement 3 times and take the average value.

[0051] 1.3 Determination of the cedrol content 1.3.1 Chromatographic conditions The chromatographic column was a Fortis XI C8 (250×4.6 mm, 5 μm); the mobile phase was acetonitrile-0.1% phosphoric acid aqueous solution (85:15); the detection wavelength was 190 nm; the column temperature was 30 ℃; the flow rate was 1.0 mL / min; and the injection volume was 10 μL.

[0052] 1.3.2 Preparation of reference solution Accurately weigh 10 mg of cedrol reference standard, place it in a 50 mL volumetric flask, add a certain amount of acetonitrile to dissolve it by sonication, and dilute to the mark to obtain a 200 μg / mL cedrol reference standard solution. Seal and store at 4 ℃ for later use.

[0053] 1.3.3 Preparation of the test solution Accurately pipette 1 mL of CE-NS suspension into a 50 mL volumetric flask, add acetonitrile, sonicate to dissolve, and dilute to the mark to obtain the CE-NS test solution.

[0054] 1.3.4 Specificity Examination Prepare negative control samples using excipients such as SDS and HPMC, following the method described in section "1.3.3". Perform HPLC analysis on cedrol reference solution, CE-NS test solution, and negative control solution according to the chromatographic conditions described in section "1.3.1". The chromatograms are shown in the figure. Figure 1 The results showed that the chromatographic peak of cedrol was not affected by other components, indicating that the method has good specificity.

[0055] 1.3.5 Examination of Linear Relationships Accurately pipette the cedrol reference solution and dilute it with acetonitrile to prepare a series of cedrol reference solutions with mass concentrations of 5, 10, 25, 50, 100, and 200 μg / mL. Perform the determination according to the chromatographic conditions described in section 1.3.1, and record the peak areas. Perform linear regression analysis with mass concentration as the abscissa (X) and peak area as the ordinate (Y), obtaining the linear regression equation Y = 551.71X + 598.12, R0. 2 =0.9998, indicating that cedrol showed good linearity in the range of 5–200 μg / mL.

[0056] 1.3.6 Precision Examination A 100 μg / mL solution of cedrol reference standard was injected and analyzed six times consecutively under the chromatographic conditions described in section 1.3.1. The results showed that the peak area of ​​cedrol was 0.62%, indicating that the instrument had good precision.

[0057] 1.3.7 Stability Assessment The CE-NS test solution was taken and measured at 0, 2, 4, 8, 12 and 24 h after preparation, respectively, according to the chromatographic conditions under section 1.3.1. The RSD of the peak area of ​​cedrol was calculated to be 1.61%, indicating that the CE-NS test solution has good stability within 24 h.

[0058] 1.3.8 Repeatability Test Six test solutions were prepared from the CE-NS suspension according to the method in section 1.3.3, and determined under the chromatographic conditions in section 1.3.1. The RSD of the mass fraction of cedrol was calculated to be 1.24%, indicating that the method has good repeatability.

[0059] 1.3.9 Recovery rate assessment Nine 0.5 mL aliquots of CE-NS suspension were placed in 25 mL volumetric flasks and divided into three groups: low, medium, and high concentrations. Cedrol reference standard was added at 80%, 100%, and 120% concentrations, respectively. The cedrol content was determined and the recovery rate was calculated under the chromatographic conditions described in section 1.3.1. The results showed that the average recovery rate of CE-NS was 100.41%, with an RSD of 0.86%, indicating that the method has high accuracy.

[0060] 1.4 Single-factor analysis of prescriptions 1.4.1 Screening of stabilizers The effects of different stabilizers on the particle size, PDI (polydispersity index), and Zeta potential of CE-NS were investigated under the following conditions: 0.5 g of cedrol, 0.1 g of stabilizer (types shown in Table 1), 6.2 g of PEG400 solvent, 93.2 g of water, a PEG400 to water weight ratio of 1:15, a final drug concentration of 5 mg / mL, a solvent dropping rate of 10 mL / min, a stirring speed of 10000 r / min, and a shearing time of 5 min. The results are shown in Table 1. The results in Table 1 show that when SDS is used in combination with HPMC as a stabilizer, the average particle size and PDI of the prepared CE-NS are smaller. Therefore, the combination of SDS and HPMC is chosen as the stabilizer for CE-NS.

[0061] Table 1. Effect of stabilizer type on particle size and PDI

[0062] 1.4.2 Screening of the SDS / HPMC ratio of the composite stabilizer The effects of different ratios of SDS and HPMC on the particle size, PDI, and Zeta potential of CE-NS were investigated under the following conditions: 0.5 g of cedrol, a weight ratio of PEG400 to water of 1:15, a final drug concentration of 5 mg / mL, a solvent dropping rate of 10 mL / min, a stirring speed of 10000 r / min, and a shearing time of 5 min. The results are shown in Table 2. Table 2 shows that as the proportion of SDS increases, the average particle size, PDI, and Zeta potential of CE-NS gradually increase. The average particle size and PDI of CE-NS prepared with an SDS:HPMC ratio of 1:1 (g / g) are the smallest. Therefore, a 1:1 weight ratio of SDS to HPMC was selected as the stabilizer for preparing CE-NS.

[0063] Table 2. Effect of SDS / HPMC stabilizer ratio on particle size and PDI

[0064] 1.4.3 Investigation into the Dosage of Composite Stabilizer The effects of the amount of composite stabilizer on the particle size, PDI, and Zeta potential of CE-NS were investigated under the following conditions: 0.5 g of cedrol, a 1:1 ratio of SDS / HPMC stabilizer, a 1:15 weight ratio of PEG400 to water, a final drug concentration of 5 mg / mL, a solvent dropping rate of 10 mL / min, a stirring speed of 10000 r / min, and a shearing time of 5 min. The results are shown in Table 3. As can be seen from the results in Table 3, the average particle size and PDI of the prepared CE-NS were minimized when the amount of composite stabilizer in the formulation was 0.3 g. Therefore, the amount of stabilizer selected was 0.3 g.

[0065] Table 3. Effect of stabilizer dosage on particle size and PDI

[0066] 1.4.4 Screening of cedrol concentration The effects of cedrol concentration on the particle size, PDI, and Zeta potential of CE-NS were investigated under the following conditions: a total amount of stabilizer SDS / HPMC of 0.3 g, a stabilizer ratio of 1:1, a PEG400 to water weight ratio of 1:15, a solvent dropping rate of 10 mL / min, a stirring speed of 10000 r / min, and a shearing time of 5 min. The results are shown in Table 4. As can be seen from the results in Table 4, with the increase of cedrol concentration, the average particle size and PDI of CE-NS generally showed a trend of first decreasing and then increasing. When the concentration of cedrol was 7.5 mg / mL, the average particle size and PDI of the prepared CE-NS were smaller. Therefore, a cedrol concentration of 7.5 mg / mL was selected.

[0067] Table 4. Effect of cedrol concentration on particle size and PDI

[0068] 1.4.5 Solvent to Antisolvent Ratio The effects of the weight ratio of PEG400 to water on the particle size, PDI, and Zeta potential of CE-NS were investigated under the following conditions: cedrol concentration of 7.5 mg / mL, stabilizer dosage of 0.3 g, stabilizer ratio of 1:1, solvent dropping rate of 10 mL / min, stirring speed of 10000 r / min, and shearing time of 5 min. The results are shown in Table 5. As can be seen from the results in Table 5, the CE-NS prepared with a PEG400 to pure water ratio of 1:12.5 had relatively small particle size and PDI. Therefore, a solvent to antisolvent ratio of 1:12.5 was chosen.

[0069] Table 5. Effect of solvent to antisolvent ratio on particle size and PDI

[0070] 1.5 Single-factor analysis of the process 1.5.1 Dropping Acceleration The concentration of cedrol was fixed at 7.5 mg / mL, the amount of stabilizer was 0.3 g, the stabilizer ratio was 1:1, the weight ratio of PEG400 to water was 1:12.5, the stirring speed was 10000 r / min, and the shearing time was 5 min. The dropping rate was investigated, and the results are shown in Table 6. Table 6 shows that when the dropping rate was 20 mL / min, the prepared CE-NS particles had smaller particle size and PDI; therefore, a dropping rate of 20 mL / min was selected.

[0071] Table 6. Effect of dropping rate on particle size and PDI

[0072] 1.5.2 Stirring speed The concentration of cedrol was fixed at 7.5 mg / mL, the amount of stabilizer was 0.3 g, the stabilizer ratio was 1:1, the weight ratio of PEG400 to water was 1:12.5, the dropping rate was 20 mL / min, and the shearing time was 5 min. The stirring speed was investigated, and the results are shown in Table 7. As shown in Table 7, the CE-NS particles with smaller particle size and PDI were obtained at a stirring speed of 10000 r / min. Therefore, a stirring speed of 10000 r / min was selected.

[0073] Table 7. Effect of stirring speed on particle size and PDI

[0074] 1.5.3 Shearing Time The concentration of cedrol was fixed at 7.5 mg / mL, the amount of stabilizer was 0.3 g, the stabilizer ratio was 1:3, the weight ratio of PEG400 to water was 1:12.5, the dropping rate was 20 mL / min, and the stirring speed was 10000 r / min. The shear time was investigated, and the results are shown in Table 8. Table 8 shows that when the shear time was 5 min, the prepared CE-NS particles had smaller particle size and PDI; therefore, a shear time of 5 min was selected.

[0075] Table 8. Effect of shear time on particle size and PDI

[0076] 1.6 Box-Behnken Response Surface Methodology for Optimizing Formulation and Process 1.6.1 Box-Behnken process factor experimental design and results For CE-NS, particle size has a significant impact on drug bioavailability and efficacy. Therefore, the Box-Behnken response surface methodology was used to optimize the preparation process and formulation of CE-NS. Based on the results of single-factor experiments, the preparation process was optimized with dropping rate (X1), stirring speed (X2), and shear time (X3) as independent variables, and average particle size (Y1) and PDI (Y2) as evaluation indicators. The levels of each factor and the experimental design and results are shown in Table 9.

[0077] Table 9. Experimental Design and Results of the Box-Behnken Process (n=3)

[0078] 1.6.2 Establishment and Significance Analysis of the Quadratic Multiple Regression Model Design-Expert 13 software was used to process the data and optimize the preparation process parameters in the response surface methodology. With X1, X2, and X3 as independent variables and Y1 and Y2 as dependent variables, a quadratic polynomial equation was fitted. The results are as follows: Y1 = 458.08 - 19.24X1 + 41.02X2 - 40.24X3 - 1.05X1X2 - 62.88X1X3 + 22.85X2X3 + 126.85X1² + 85.92X2² + 233.90X3², R0 2 =0.9800; Y2=0.2342+0.0014X1-0.0026X2-0.0032X3-0.0048X1X2-0.0075X1X3+0.0040X2X3+0.0140X1²+0.0130X2²+0.0243X3², R 2=0.9454.

[0079] For the Y1 model equation, P < 0.0001, indicating extreme significance, while the lack-of-fit term P = 0.1041 > 0.05, indicating no significance. For the Y2 model equation, P = 0.0012 < 0.05, indicating significance, while the lack-of-fit term P = 0.2088 > 0.05, indicating no significance. This demonstrates that the model has a good fit and can be used for optimizing the CE-NS preparation process.

[0080] 1.6.3 Response Surface Analysis and Optimization Results Using X1, X2, and X3 as independent variables and Y1 and Y2 as dependent variables, with one independent variable fixed, response surface plots were generated using Design-Expert 13 software. Y1 is the main indicator for evaluating nanoparticles. Ideal nanoparticles should have the smallest possible particle size to maximize their size effect, be uniformly distributed, have a high drug loading capacity, and be simple to prepare. Therefore, the optimal preparation process for CE-NS is a dropping rate of 20.355 mL / min, a stirring speed of 9987.2 r / min, and a shearing time of 5.173 min.

[0081] 1.6.4 Box-Behnken Prescription Factors Experiment Design and Results The formulation used SDS dosage (X1), HPMC dosage (X2), cedrol mass concentration (X3), and solvent-to-antisolvent ratio (X4) as independent variables, and average particle size (Y1) and PDI (Y2) as evaluation indicators. The levels of each factor and the experimental design and results are shown in Table 10.

[0082] Table 10. Box-Behnken Prescription Trial Design and Results (n=3)

[0083] 1.6.5 Establishment and Significance Analysis of the Quadratic Multiple Regression Model Design-Expert 13 software was used to process the data and optimize the preparation parameters in the response surface methodology. A quadratic polynomial equation was fitted with X1, X2, X3, and X4 as independent variables and Y1 and Y2 as dependent variables. The results are as follows: Y1 = 438.77 - 106.22X1 + 94.07X2 + 27.23X3 + 54.43X4 - 58.43X1X2 - 24.00X1X3 - 20.12X1X4 - 19.95X2X3 + 129.38X2X4 + 196.28X3X4 + 296.67X1 2 +176.80X2 2 +263.46X3 2+310.91X4 2 ;Y2=0.2335-0.0341X1-0.0089X2-0.0004X3+0.0091X4+0.0130X1X2-0.0097X1X3-0.0320X 1X4-0.0110X2X3+0.0083X2X4+0.0175X3X4+0.0600X1²-0.0068X2²+0.0202X3²+0.0330X4².

[0084] For the Y1 model equation, P < 0.0001, indicating extreme significance, while the lack-of-fit term P = 0.0562 > 0.05, indicating no significance. For the Y2 model equation, P = 0.0002 < 0.05, indicating significance, while the lack-of-fit term P = 0.2653 > 0.05, indicating no significance. This demonstrates that the model has a good fit and can be used for optimizing the formulation of CE-NS preparations.

[0085] 1.6.6 Response Surface Analysis and Optimization Results Using X1, X2, X3, and X4 as independent variables and Y1 and Y2 as dependent variables, with one independent variable fixed, response surface plots were generated using Design-Expert 13 software. The optimal formulation for CE-NS was: 172.776 mg SDS, 133.190 mg HPMC, 0.745% cedrol, and a weight ratio of PEG400 to aqueous phase of 1:12.306.

[0086] 1.7 Determination and Validation of Optimal Formulation Process Considering practical operability, the optimized formulation process validation was slightly adjusted as follows: cedrol concentration was 0.75%, SDS dosage was 170 mg, HPMC dosage was 130 mg, and the weight ratio of PEG400 to aqueous phase was 1:12; the dropping rate was 20 mL / min, the stirring speed was 10000 r / min, and the shearing time was 5 min. Three batches of CE-NS were prepared on average, and the particle size distribution (PDI) was measured for each batch. The deviation from the predicted value was calculated using the formula: Deviation (%) = (Actual value - Predicted value) / Predicted value × 100%. The results showed that the average particle size of the cedrol nanosuspension was 434.47 ± 9.90 nm, and the PDI was 0.23 ± 0.005. The predicted particle size was 420.52 nm, and the deviation from the actual value was 3.32%, indicating that the model has good reliability and predictive ability, and can be used for formulation process optimization.

[0087] 1.8 Characterization of cedrol nanosuspension 1.8.1 Determination of Particle Size Distribution and Zeta Potential Three batches of cedrol nano-suspension were taken and analyzed according to the method described in section "1.2". The particle size distribution is shown in the figure. Figure 2 As shown, the distribution range is 434.47±9.90 nm, the PDI value is 0.23±0.005, and the Zeta potential value is -15.2±1.15 mV. 1.8.2 Stability Study of CE-NS The prepared CE-NS was sampled at predetermined time points under the following conditions: high temperature (40 ℃), light intensity (4500±500) lx, room temperature, and 4 ℃. The cedrol content was determined by high performance liquid chromatography (HPLC), and the particle size, PDI, and potential were measured using a laser particle size analyzer. The results are shown in Tables 11, 12, 13, and 14, respectively. The preliminary stability of CE-NS was investigated to predict its storage conditions. It was found that under high temperature, strong light, room temperature, and 4 ℃ conditions, the redispersibility of the nano-suspension was minimally affected after 3 months, and the color showed no significant change. However, under high temperature conditions, the particle size and PDI increased with prolonged storage time, indicating that the nano-suspension has good stability when stored at room temperature.

[0088] Table 11. Results of High Temperature Stability Test

[0089] Table 12. Results of the strong light stability test

[0090] Table 13. Results of Room Temperature Stability Test

[0091] Table 14. Results of the 4℃ stability test

[0092] Example 2: Formulation and preparation process of cedrol nano-suspension Prescription 1: Cedarol 0.75 g, SDS 0.17 g, HPMC 0.13 g, PEG400 7.60 g, water 91.35 g, total 100 g.

[0093] The preparation method includes the following steps: Step (1): Accurately weigh 0.75g of cedrol and place it in a beaker equipped with a magnetic stir bar. Add 7.60g of PEG400 and place it in a constant temperature water bath at 90±2℃. Continue stirring until the cedrol is completely dissolved to form a clear, homogeneous, and transparent drug solution, which is used as the drug phase. Allow it to cool at room temperature for later use. Step (2): Add 0.17g SDS and 0.13g HPMC to 91.35g of purified water in sequence. Stir at 300~500 r / min for 10~20 min at room temperature until SDS and HPMC are completely dissolved to form a homogeneous, colorless and transparent aqueous solution. Use this as the aqueous phase and place it in an ice-water bath for later use. Step (3): The drug phase in step (1) is added dropwise to the aqueous phase in step (2) at a rate of 20 mL / min using a peristaltic pump. During the dropwise addition, a high-speed shear dispersion emulsifier is used to continuously shear and emulsify at a speed of 10,000 r / min. After the drug phase is completely added, the shearing is continued at the same speed for 5 min to obtain the cedrol nano suspension of the present invention.

[0094] Example 3: Formulation and preparation process of cedrol nano-suspension Prescription 2: Cedarol 0.50 g, SDS 0.17 g, HPMC 0.13 g, PEG400 7.63 g, water 91.57 g, total 100 g.

[0095] The preparation method includes the following steps: Step (1): Accurately weigh 0.50g of cedrol and place it in a beaker equipped with a magnetic stir bar. Add 7.63g of PEG400 and stir continuously in a constant temperature water bath at 90±2℃ until the cedrol is completely dissolved to form a clear, homogeneous, and transparent drug solution. This solution is used as the drug phase and cooled to room temperature for later use. Step (2): Add 0.17g SDS and 0.13g HPMC to 91.57g purified water in sequence. Stir at 300~500 r / min for 10~20 min at room temperature until SDS and HPMC are completely dissolved to form a homogeneous, colorless and transparent aqueous solution. Use this as the aqueous phase and place it in an ice-water bath for later use. Step (3): The drug phase in step (1) is added dropwise to the aqueous phase in step (2) at a rate of 18 mL / min using a peristaltic pump. During the dropwise addition, a high-speed shear dispersion emulsifier is used to continuously shear and emulsify at a speed of 9000 r / min. After the drug phase is added, the shearing is continued at the same speed for 4 min to obtain the cedrol nano suspension of the present invention.

[0096] Example 4: Formulation and preparation process of cedrol nano-suspension Prescription 3: Cedarol 1.0 g, SDS 0.17 g, HPMC 0.13 g, PEG400 7.59 g, water 91.11 g, total 100 g.

[0097] The preparation method includes the following steps: Step (1): Accurately weigh 1.0g of cedrol and place it in a beaker equipped with a magnetic stir bar. Add 7.59g of PEG400 and place it in a constant temperature water bath at 90±2℃. Continue stirring until the cedrol is completely dissolved to form a clear, homogeneous, and transparent drug solution, which is used as the drug phase. Allow it to cool at room temperature for later use. Step (2): Add 0.17g SDS and 0.13g HPMC to 91.11g of purified water in sequence. Stir at 300~500 r / min for 10~20 min at room temperature until SDS and HPMC are completely dissolved to form a homogeneous, colorless and transparent aqueous solution. Use this as the aqueous phase and place it in an ice-water bath for later use. Step (3): The drug phase in step (1) is added dropwise to the aqueous phase in step (2) at a rate of 22 mL / min using a peristaltic pump. During the dropwise addition, a high-speed shear dispersion emulsifier is used to continuously shear and emulsify at a speed of 11000 r / min. After the drug phase is added, the shearing is continued at the same speed for 6 min to obtain the cedrol nano suspension of the present invention.

[0098] Example 5: Formulation and preparation process of cedrol nano-suspension Prescription 4: Cedarol 0.75 g, SDS 0.15 g, HPMC 0.15 g, PEG400 9.00 g, water 89.95 g, total 100 g.

[0099] The preparation method is the same as in Example 2.

[0100] Example 6: Formulation and preparation process of cedrol nano-suspension Prescription 5: Cedarol 0.75 g, SDS 0.18 g, HPMC 0.12 g, PEG400 6.18 g, water 92.77 g, total 100 g.

[0101] The preparation method is the same as in Example 2.

[0102] Example 7: Formulation and preparation process of cedrol nano-suspension Prescription 6: Cedarol 0.50 g, SDS 0.15 g, HPMC 0.15 g, PEG400 9.02 g, water 90.18 g, total 100 g.

[0103] The preparation method is the same as in Example 2.

[0104] Example 8: Formulation and preparation process of cedrol nano-suspension Prescription 7: Cedarol 0.50 g, SDS 0.18 g, HPMC 0.12 g, PEG400 6.20 g, water 93.00 g, total 100 g.

[0105] The preparation method is the same as in Example 2.

[0106] Example 9: Formulation and preparation process of cedrol nano-suspension Prescription 8: Cedarol 1.0 g, SDS 0.15 g, HPMC 0.15 g, PEG400 8.97 g, water 89.73 g, total 100 g.

[0107] The preparation method is the same as in Example 2.

[0108] Example 10: Formulation and preparation process of cedrol nano-suspension Prescription 9: Cedarol 1.0 g, SDS 0.18 g, HPMC 0.12 g, PEG400 6.17 g, water 92.53 g, total 100 g.

[0109] The preparation method is the same as in Example 2.

[0110] Example 11: In vitro transdermal experiment of cedrol nanosuspension 11.1 The transdermal behavior and intradermal retention of the cedrol nanosuspension (CE-NS) prepared in Example 2 of this invention were evaluated using the Franz diffusion cell method, with a free cedrol solution of the same mass concentration (Free-CE) as a control. Pig skin with the keratin layer facing upwards was fixed between the supply and receiving cells, with an effective diffusion area of ​​1.77 cm². 2 The receiving cell had a volume of 12 mL, used PBS solution as the receiving fluid, and was kept in a constant temperature water bath at (32±2)℃ with a rotation speed of 600 rmin. -1 0.5 mL of CE-NS and Free-CE solutions were evenly applied to the epidermal layer of the skin. At 1, 2, 4, 6, 8, 10, 12, and 24 hours, 2 mL of the receiving solution was collected, and an equal volume of fresh receiving medium was added. The collected receiving solutions were filtered through a 0.22 μm microporous membrane, and the cedrol content was determined by HPLC. The cumulative permeation per unit area was calculated according to the formula (…). ).

[0111]

[0112] This represents the mass concentration of the drug in the receiving solution up to the nth sampling point, where V is the total volume of the receiving solution. For the first The mass concentration of the drug in the receiving solution during the second sampling. Let A be the volume of each sample taken, and let A be the effective penetration area.

[0113] The results showed that CE was not detected in either CE-NS or Free-CE in the receiving solution after 24 h, indicating that the drug had a good cumulative effect on the skin.

[0114] Skin retention determination: After the transdermal test, the skin was removed, and any remaining drug on the surface was rinsed off with physiological saline. The surface moisture was then blotted dry with filter paper, and the skin was minced. 3 mL of anhydrous ethanol was added, and the mixture was sheared for 30 min, vortexed for 5 min, and sonicated for 10 min to fully extract the drug. The resulting sample was centrifuged at 4000 r / min for 10 min, and the supernatant was filtered through a 0.22 μm filter membrane and subjected to HPLC to determine the cedrol content. The formula for calculating skin retention is as follows: Where Cm is the extract concentration, V is the extract volume, and A is the transdermal penetration area. Results are shown below. Figure 3 The skin retention of CE-NS was 4.1 times that of Free-CE, and the difference was statistically significant (P < 0.05).

[0115] 11.2 Attenuated Total Reflectance-Fourier Transform Infrared Spectroscopy (ATR-FTIR) Characterization of Pigskin Changes in the stratum corneum and keratin of porcine skin after transdermal treatment were characterized and analyzed using ATR-FTIR. Following the method described in section "2.9", the skin was treated with 0.5 mL of PBS solution, CE-NS solution, and Free-CE solution, respectively, with the PBS solution group serving as a blank control. 24 h after transdermal treatment, the porcine skin was removed. The surface of the skin was gently rinsed with physiological saline to remove any remaining sample, then blotted dry with filter paper. The skin was then dried in a desiccator for 72 h with the stratum corneum facing upwards for ATR-FTIR testing. The ATR-FTIR testing conditions were as follows: the porcine skin was placed face down on a ZnSe ATR crystal of the infrared spectrometer, pressed firmly with a flat-head indenter to prevent air ingress, and the temperature was maintained between 4000 and 650 cm⁻¹. -1 100 consecutive scans within the wavenumber range, with a resolution of 2cm. -1 After completion, Origin 2022 was used to process the experimental data. Results are shown below. Figure 4 The asymmetric stretching vibration peak (VasCH2) and symmetric stretching vibration peak (VsCH2) of CH2 in the intercellular lipid molecules of the stratum corneum are located at 2920 cm⁻¹. -1 and 2850 cm -1 Left and right. In the control group, the spectrum showed that VasCH2 in the intercellular lipid molecules of the stratum corneum was located at 2921.38 cm⁻¹. -1VsCH2 is located at 2851.99 cm. -1 After treatment with Free-CE and CE-NS, VasCH2 and VsCH2 were observed to shift to higher wavenumbers, reaching 2922.73 cm⁻¹. -1 / 2852.77 cm -1 and 2924.07cm -1 / 2853.73 cm -1 When the CH2 stretching vibration shifts to a higher wavenumber, it indicates that the lipid structure of the stratum corneum is disturbed and transforms from an orthorhombic crystal structure to a liquid crystal state, enhancing the intercellular lipid molecular flow phase.

[16] Furthermore, the characteristic peaks of amide I and amide II, attributed to the secondary structure of the keratin helix, are located at 1640 cm⁻¹. -1 and 1540 cm -1 Approximately 1629.88 cm after Free-CE treatment. -1 and 1545.01 cm -1 The absorption bands at these locations change to 1634.70 cm⁻¹. -1 and 1543.08 cm -1 After CE-NS treatment, the result became 1639.52 cm. -1 and 1543.08 cm -1 This indicates that the secondary structure of proteins in the skin has changed.

[17] In summary, both Free-CE and CE-NS treatments can reduce the stratum corneum barrier function of pigskin, but the effect of CE-NS as described in this invention is more significant.

[0116] 11.3 Observation of hair follicles by scanning electron microscopy (SEM) The microstructure of hair follicles treated with CE nanocrystals was visualized using SEM to investigate the follicle targeting ability of the nanocrystals. In vitro transdermal experiments were conducted according to the experimental procedures described in Part 1 of Example 11 above. The experiment was divided into two groups: a PBS blank group and a CE-NS group. A Franz diffusion apparatus was used. Skin samples were removed 24 h after drug administration. Residual preparations were first removed with physiological saline, then blotted dry with filter paper. The skin samples were then cut with a scalpel blade in a direction nearly perpendicular to the surface. The skin was dried in a desiccator for 72 h. The microstructure of hair follicles treated with the nano-suspension was observed using scanning electron microscopy to observe the nanocrystals within the hair follicles. Results are shown below. Figure 5As shown in Figures C and D, many nanocrystals can be clearly observed around the hair follicles. However, no CE nanocrystal structure was found around the hair follicles in the control group treated with PBS solution (Figures A and B). This indicates that local application of nanosuspension can increase drug penetration into the area around the hair follicles, providing strong evidence for the mechanism of CE-NS targeting hair follicles.

[0117] Example 12, Pharmacodynamic Study 12.1 Grouping, establishment, and administration of a mouse model of androgenetic alopecia Sixty healthy SPF-grade C57BL / 6 male mice weighing 20-22 g were acclimatized for one week and randomly divided into six groups: a blank control group (Control), a model group (Model), a positive control group (Minoxidil), a blank nanosuspension group (Blank-NS), a free cedrol group (Free CE), and a cedrol nanosuspension group (CE-NS), with 10 mice in each group. The fur on the back of each mouse was shaved in a 2 cm × 2 cm area using a pet shaver to avoid skin damage. Depilatory cream was then applied for 1-2 minutes to remove any remaining hair, and then wiped off with warm water. Except for the blank control group, all other groups of mice received subcutaneous injections of diluted testosterone propionate at a dose of 5 mg·kg⁻¹ for 21 consecutive days. Thirty minutes after modeling each day, 1 mL of physiological saline was applied to the blank control group and the model group, 0.15 mL of minoxidil solution was applied to the positive control group, and the corresponding drug solutions were applied to the blank nanosuspension group, the free cedrol group, and the cedrol nanosuspension group, respectively. The dosage was 1 mL per mouse, and this treatment was repeated for 21 consecutive days. The weight of the mice was measured and recorded every other day. Using the weight of each mouse as a reference, the presence of drug toxicity during the experiment was observed. The results are shown in […]. Figure 6 Throughout the experiment, the body weight of mice in each group showed an increasing trend with prolonged feeding time. On day 21, compared with the control group, the body weight of mice in each group receiving daily testosterone propionate was significantly increased, confirming the androgenic effect of testosterone propionate in mice. Compared with the model group, there were no significant differences in body weight among the minoxidil group, the control group, the free cedrol group, and the cedrol nanosuspension group, indicating that application of CE-NS to mice was non-toxic.

[0118] 12.2 Hair growth in mice The hair growth on the backs of all groups of mice was observed daily. Photos of the mouse back skin were taken on days 1, 7, 14, and 21 of the experiment, and the time when the skin began to darken and hair regrowth began was recorded. The results of hair growth in the bald areas of the mice are shown below. Figure 7 The coverage rate of new hair growth on day 21 was quantified using ImageJ software, and the results are shown in [Figure Number]. Figure 8Throughout the process, daily observation and recording were required of the mice's backs after topical application of the medication to prevent redness, swelling, exudation, ulceration, dryness, and peeling. Results showed that on day 1 of modeling, the skin on the backs of all groups of mice was pink, indicating that the hair follicles were in the resting phase. On day 7 of administration, the skin on the backs of mice in the Control group was grayish-black with no hair growth, indicating that the hair follicle growth cycle had begun; the skin color on the backs of the Minoxidil and CE-NS groups was similar to the control group, while the skin on the backs of the Model mice remained predominantly pink, revealing that their hair follicles were still in the resting phase. On day 14 of administration, the hair on the backs of mice in the Control group showed significant growth, with short and dense hair; the skin on the backs of mice in the Blank-NS and Free-CE groups was grayish-black, but no hair growth was observed; the backs of mice in the Minoxidil and CE-NS groups were grayish-black, and the newly grown hair was distributed in a "patchy" pattern, but the CE-NS group had a larger coverage area than the Minoxidil group, indicating that cedrol can accelerate the entry of hair follicles into the growth phase. On day 21 of drug administration, the hair loss area in the Control group was almost completely covered; the Model and Blank-NS groups showed sparse hair overall, with some areas showing no hair growth; the Free CE group still had some areas with sparse hair growth; the Minoxidil and CE-NS groups showed significant hair growth, almost completely covering the affected area, and the CE-NS group showed a better effect on promoting hair regeneration compared to the Minoxidil and Free CE groups. Hair coverage on day 21 showed that compared to the control group, the hair coverage in the model group was significantly lower, while the hair coverage in the positive control group, free cedrol group, and cedrol nanosuspension group was significantly higher than that in the model group. In conclusion, the androgenic alopecia mouse model was successfully established using subcutaneous injection of testosterone propionate. Cedarol has a certain alleviating effect on androgenic alopecia and can promote hair growth. Furthermore, the nanosuspension delivery system can significantly promote the transdermal penetration of active substances, increasing the therapeutic effect.

[0119] 12.3 Measurement of weight and length of new hair growth Mice were sacrificed on day 21, and newly grown hair on the backs of the mice was collected and weighed. The length of the newly grown hair was also measured using calipers. The results are shown in Table 15. Compared with the Control group, the length and weight of newly grown hair in the Model group were significantly reduced. In terms of hair length, compared with the Model group, the length of newly grown hair in all treatment groups was significantly increased. In terms of hair weight, compared with the Model group, the weight of newly grown hair in the CE-NS group (25.03±1.08mg) was significantly higher than that in other groups. In summary, visual observation of hair growth and quantitative analysis of hair coverage showed that, compared with the Model group, Minoxidil, Free-CE, and CE-NS all significantly promoted hair regeneration. Among them, the CE-NS described in this invention had the best effect, with a hair coverage of 90.19%, a newly grown hair length of (4.94±0.16) mm, and a weight of (25.03±1.08) mg. The measurement of hair length and weight further proved the effectiveness of the CE-NS described in this invention in promoting hair growth. Furthermore, although Free-CE has shown some hair growth promoting effect compared to the Model group, confirming the intrinsic biological activity of cedrol, the therapeutic effect of CE-NS described in this invention is superior to other groups, including superior to the positive drug minoxidil.

[0120] Table 15. Length and weight of newly grown hair in each group of mice

[0121] 12.4 Observation of skin tissue pathological changes by hematoxylin-eosin (HE) staining After euthanizing mice, skin from the experimental area was excised and fixed in 4% paraformaldehyde tissue fixative for at least 24 hours. Following dehydration, the tissue was cleared, embedded in paraffin, sectioned, dehydrated and hydrated, stained with hematoxylin, subjected to differentiation blue staining, eosin staining, cleared again, and mounted. Observations and records were made under an optical microscope. HE staining results are shown in [Figure number missing]. Figure 9 On day 21 of the experiment, the morphology of hair follicles in mouse skin tissue sections revealed that, compared with the control group, the Model group mice had thinner skin, fewer hair follicles, and most hair follicles were atrophied and had not yet entered the proliferative phase, indicating that hair growth was inhibited by testosterone propionate and that the hair follicles were still in the resting phase. Mice treated with Free CE, CE-NS, and Minoxidil showed a significant increase in the number of regenerated hair follicles and were in the growth phase. However, compared with the Free CE and Minoxidil groups, the CE-NS group mice had more hair follicles in their skin tissue, with intact hair follicle structure, larger hair bulbs, mature hair shafts, and significantly increased skin thickness, indicating that the hair follicles in the CE-NS group mice had entered the growth phase. This demonstrates the superiority of this nano-suspension delivery system in promoting hair regeneration.

[0122] 12.5 Immunohistochemical staining (IHC) experiment To investigate the effects of cedrol on hair follicle proliferation and apoptosis, paraffin-embedded skin tissue sections were prepared. These sections were then stained with Androgen receptor (AR) antibody, Ki67 antibody, and β-catenin antibody, respectively, using immunohistochemical staining. The sections were then observed and photographed using an optical microscope to assess the effect on hair follicle microenvironment remodeling. Results are shown below. Figure 10 Compared with the control group, the expression of AR protein in the skin of mice in the model group was significantly increased, indicating that there is only a few or no expression of androgen receptors around normal hair follicles. This suggests that when testosterone propionate is injected subcutaneously into mice, androgens may competitively bind to AR, inducing an increase in AR expression around hair follicles. After CE-NS treatment, AR protein levels were downregulated compared to the Model group, indicating that cedrol can regulate AR expression levels. Ki67 expression was very low in the Model group, indicating that hair follicles had entered the resting phase or their proliferative activity was inhibited. Compared to the Model group, Ki67 expression was increased to varying degrees in all treatment groups (Minoxidil, Free-CE, and CE-NS), indicating that minoxidil and cedrol promoted the transition of the hair follicle growth cycle and the proliferation of cells related to hair regeneration, and alleviated AGA symptoms. The positive expression of β-catenin in the Model and Blank-NS groups was significantly reduced compared to the Control group, and the positive staining area was significantly smaller. Compared to other treatment groups, the positive expression of β-catenin in the CE-NS group was significantly higher, suggesting that it may promote hair regeneration by activating the Wnt / β-catenin pathway. In summary, immunohistochemical results show that cedrol nanosuspension (CE-NS) regulates the hair follicle growth cycle and promotes hair stem cell proliferation and hair regeneration by downregulating AR expression levels and upregulating Ki67 and β-catenin expression levels.

[0123] Comparative Example 1: This invention studies the method for determining the content of cedrol in cedrol nano-suspension using HPLC under different detection conditions.

[0124] (1) Detection conditions for cedrol: Instrument: Shimadzu Column: Diamonsil Diamond II C18 (250 × 4.6 mm, 5 μm), mobile phase A: acetonitrile, mobile phase B: 1% phosphoric acid aqueous solution, gradient elution.

[0125] Flow rate: 1.0 mL / min, column temperature: 25℃, wavelength: 210 nm, injection volume: 10 μL (2) Results: We performed liquid chromatography analysis according to the method of Sichuan Weikeqi Biotechnology Co., Ltd. for cedrol standard, and the results are as follows. Figure 11 As shown, no drug peak was observed. Considering it was a single component, isocratic elution would be more convenient. Furthermore, conventional C18 columns are only suitable for pH ranges of 2.0–7.5, while the pH of 1% phosphoric acid aqueous solution is too low, which would reduce the column's lifespan with prolonged use. Therefore, the mobile phase ratio was subsequently adjusted to acetonitrile:0.1% phosphoric acid aqueous solution (90:10) for injection analysis.

[0126] Comparative Example 2: This invention studies the method for determining the content of cedrol in cedrol nano-suspension using HPLC under different detection conditions.

[0127] (1) Detection conditions for cedrol: Instruments: Shimadzu; Column: Diamonsil C18 II (250×4.6 mm, 5 μm); Mobile phase A: acetonitrile; Mobile phase B: 0.1% phosphoric acid water; Isocratic elution: A phase: B phase (90:10); Flow rate: 1.0 mL / min; Column temperature: 25℃; Wavelength: 210 nm; Injection volume: 10 μL.

[0128] (2) Results: such as Figure 12 As shown, around 7.5–8 min, the cedrol reference solution showed several more peaks than the blank solution, presumably indicating that this was the main peak of cedrol. Subsequent analysis with increased injection volume and cedrol concentration revealed no increase in peak intensity or a linear relationship. A solution of appropriate concentration was prepared by dissolving cedrol reference in acetonitrile, using acetonitrile as a blank control. Ultraviolet (UV) scanning was performed in the 190–400 nm range using UV spectrophotometry, and the results are shown below. Figure 13 As shown, the maximum absorption peak of cedrol is located at 190 nm. At this wavelength, the solvent has no effect on the absorption of cedrol. Therefore, we tried to use 190 nm as the detection wavelength for cedrol injection analysis.

[0129] Comparative Example 3: This invention studies the method for determining the content of cedrol in cedrol nano-suspension using HPLC under different detection conditions.

[0130] (1) Detection conditions of cedrol: Instrument: Shimadzu, chromatographic column: Diamonsil Diamond II C18 (250×4.6 mm, 5 μm), mobile phase A: acetonitrile, mobile phase B: 0.1% phosphoric acid water, isocratic elution: phase A: phase B (85:15), flow rate: 1.0 mL / min, column temperature: 25℃, wavelength: 190nm / 210nm, injection volume: 10 μL.

[0131] (2) Results: The results are as follows Figure 14 As shown, the inventors discovered that when the detection wavelength was adjusted to 190 nm, a distinct cedrol chromatographic peak appeared at approximately 8 minutes; however, this peak was absent when the detection wavelength was 210 nm. Furthermore, the peak areas measured from different concentrations of cedrol reference solutions showed a good linear relationship. Therefore, the detection wavelength of 190 nm was ultimately determined. Subsequently, considering that cedrol is a terpene compound and is more suitable for C8 chromatographic columns, the separation effects of C8 and C18 columns on cedrol were compared.

[0132] Comparative Example 4: This invention studies the method for determining the content of cedrol in cedrol nano-suspension using HPLC under different detection conditions.

[0133] (1) Detection conditions for cedrol: Instruments: Shimadzu; Column: Diamonsil Diamond II C18 (250×4.6 mm, 5 μm) / Fortis XIC8 (250×4.6 mm, 5 μm); Mobile phase A: acetonitrile; Mobile phase B: 0.1% phosphoric acid water; Isocratic elution: A phase: B phase (85:15); Flow rate: 1.0 mL / min; Column temperature: 25℃; Wavelength: 190 nm; Injection volume: 10 μL.

[0134] (2) Results: such as Figure 15 As shown, the peak intensity using the C8 column was significantly higher than that using the C18 column, and the elution time was earlier. Therefore, the C8 column was chosen for analysis. Subsequently, considering the influence of high summer temperatures, the column temperature could not be maintained at 25℃, so the column temperature was adjusted to 30℃ for injection analysis.

[0135] Comparative Example 5: This invention studies the method for determining the content of cedrol in cedrol nano-suspension using HPLC under different detection conditions.

[0136] (1) Detection conditions of cedrol: Instrument: Shimadzu, chromatographic column: Fortis XI C8 (250×4.6 mm, 5 μm), mobile phase A: acetonitrile, mobile phase B: 0.1% phosphoric acid water, isocratic elution: phase A: phase B (85:15), flow rate: 1.0 mL / min, column temperature: 25℃ / 30℃, wavelength: 190nm, injection volume: 10 μL.

[0137] (2) Results: such as Figure 16 As shown, when the column temperature is adjusted to 30℃, the peak time and response intensity are basically unchanged compared to 25℃. Therefore, the column temperature was finally determined to be 30℃.

[0138] Conclusion: The chromatographic column was a Fortis XI C8 (250×4.6 mm, 5 μm); the mobile phase was acetonitrile-0.1% phosphoric acid aqueous solution (85:15); the detection wavelength was 190 nm; the column temperature was 30℃; the flow rate was 1.0 mL / min; and the injection volume was 10 μL.

[0139] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0140] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A cedrol nano-suspension, characterized in that, According to the weight percentage, each 100 parts by weight contains: 0.5 to 1.0 parts by weight of cedrol, 0.3 parts by weight of stabilizer, 6.17 to 9.02 parts by weight of solvent, and 89.73 to 93.00 parts by weight of antisolvent.

2. The cedrol nano-suspension according to claim 1, characterized in that, The stabilizer is selected from at least one of SDS, PVPK30, poloxamer 407, HPMC, Tween-80, and sodium carboxymethyl cellulose; preferably, the stabilizer is at least one of SDS, poloxamer 407, Tween-80, and HPMC; more preferably, the stabilizer is at least one of SDS and HPMC; and even more preferably, the stabilizer is a combination of SDS and HPMC. Optionally, the solvent is selected from: polyethylene glycol 400; Optionally, the antisolvent is selected from: water.

3. The cedrol nano-suspension according to claim 2, characterized in that, The stabilizer is a combination of SDS and HPMC in a weight ratio of (0.5~2.0):1; preferably, the stabilizer is a combination of SDS and HPMC in a weight ratio of (1.0~1.5):1; more preferably, the stabilizer is a combination of SDS and HPMC in a weight ratio of 1.31:

1. Optionally, the weight ratio of the solvent to the antisolvent is 1:(10~15); preferably, the weight ratio of the solvent to the antisolvent is 1:

12.

4. The cedrol nano-suspension according to claims 1-3, characterized in that, According to the weight parts, each 100 parts of its formula contains: 0.5~1.0 parts of cedrol, 0.15~0.18 parts of SDS, 0.12~0.15 parts of HPMC, 6.17~9.02 parts of PEG400, and 89.73~93.00 parts of water.

5. The cedrol nano-suspension according to claim 4, characterized in that, Based on parts by weight, each 100 parts by weight contains: 0.75 parts by weight of cedrol, 0.17 parts by weight of SDS, 0.13 parts by weight of HPMC, 7.60 parts by weight of PEG400, and 91.35 parts by weight of water; Optionally, per 100 parts by weight, it contains: 0.75 parts by weight of cedrol, 0.15 parts by weight of SDS, 0.15 parts by weight of HPMC, 9.00 parts by weight of PEG400, and 89.95 parts by weight of water; Optionally, per 100 parts by weight, it contains: 0.75 parts by weight of cedrol, 0.18 parts by weight of SDS, 0.12 parts by weight of HPMC, 6.18 parts by weight of PEG400, and 92.77 parts by weight of water; Optionally, per 100 parts by weight, it contains: 0.50 parts by weight of cedrol, 0.17 parts by weight of SDS, 0.13 parts by weight of HPMC, 7.63 parts by weight of PEG400, and 91.57 parts by weight of water; Optionally, per 100 parts by weight, it contains: 0.50 parts by weight of cedrol, 0.15 parts by weight of SDS, 0.15 parts by weight of HPMC, 9.02 parts by weight of PEG400, and 90.18 parts by weight of water; Optionally, per 100 parts by weight, it comprises: 0.50 parts by weight of cedrol, 0.18 parts by weight of SDS, 0.12 parts by weight of HPMC, 6.20 parts by weight of PEG400, and 93.00 parts by weight of water; Optionally, per 100 parts by weight, it contains: 1.0 part by weight of cedrol, 0.17 parts by weight of SDS, 0.13 parts by weight of HPMC, 7.59 parts by weight of PEG400, and 91.11 parts by weight of water; Optionally, per 100 parts by weight, it contains: 1.0 part by weight of cedrol, 0.15 part by weight of SDS, 0.15 part by weight of HPMC, 8.97 parts by weight of PEG400, and 89.73 parts by weight of water; Optionally, each 100 parts by weight contains: 1.0 part by weight of cedrol, 0.18 parts by weight of SDS, 0.12 parts by weight of HPMC, 6.17 parts by weight of PEG400, and 92.53 parts by weight of water.

6. The cedrol nano-suspension according to claims 1-5, characterized in that, Its preparation method includes the following steps: Step (1): Accurately weigh cedrol and place it in a beaker equipped with a magnetic stir bar. Add PEG400 and stir continuously in a constant temperature water bath at 90±2℃ until cedrol is completely dissolved to form a clear, homogeneous, and transparent drug solution, which is used as the drug phase. After cooling at room temperature, it is ready for use. Step (2): Add SDS and HPMC to purified water in sequence, and stir at 300~500 r / min for 10~20 min at room temperature until SDS and HPMC are completely dissolved to form a homogeneous, colorless and transparent aqueous solution, which is used as the aqueous phase and placed in an ice-water bath for later use. Step (3): The drug phase from step (1) is added dropwise to the aqueous phase from step (2) at a rate of 18 mL / min to 22 mL / min using a peristaltic pump. During the dropwise addition, a high-speed shear dispersion emulsifier is used to continuously shear and emulsify at a speed of 9000 r / min to 11000 r / min. After the drug phase has been added, the shearing is continued at the same speed for 4 to 6 minutes to obtain the cedrol nano-suspension of the present invention.

7. The cedrol nano-suspension according to claims 1-6, characterized in that, Its preparation method includes the following steps: Step (1): Accurately weigh 0.75g of cedrol and place it in a beaker equipped with a magnetic stir bar. Add 7.60g of PEG400 and place it in a constant temperature water bath at 90±2℃. Continue stirring until the cedrol is completely dissolved to form a clear, homogeneous, and transparent drug solution, which is used as the drug phase. Allow it to cool at room temperature for later use. Step (2): Add 0.17g SDS and 0.13g HPMC to 91.35g of purified water in sequence. Stir at 300~500 r / min for 10~20 min at room temperature until SDS and HPMC are completely dissolved to form a homogeneous, colorless and transparent aqueous solution. Use this as the aqueous phase and place it in an ice-water bath for later use. Step (3): The drug phase in step (1) is added dropwise to the aqueous phase in step (2) at a rate of 20 mL / min using a peristaltic pump. During the dropwise addition, a high-speed shear dispersion emulsifier is used to continuously shear and emulsify at a speed of 10,000 r / min. After the drug phase is completely added, the shearing is continued at the same speed for 5 min to obtain the cedrol nano suspension of the present invention.

8. A method for determining the cedrol content in the cedrol nanosuspension prepared according to claims 1-7, characterized in that, The method described above utilizes high-performance liquid chromatography (HPLC) combined with an ultraviolet detector for determination, wherein... (1) The chromatographic conditions are as follows: the chromatographic column is a C8 alkyl-bonded silica reversed-phase column; the mobile phase consists of acetonitrile-0.1% phosphoric acid aqueous solution; the detection wavelength is 190 nm ± 2 nm; the column temperature is 25~35℃; the flow rate is 0.8~1.2 mL / min; and the injection volume is 5~20 μL. (2) Preparation of solutions: Prepare cedrol reference solution, CE-NS test solution and negative sample solution respectively; (3) Determination: Inject the reference solution, test solution and negative sample solution from step (2) above into the high performance liquid chromatograph, and perform the determination according to the chromatographic conditions of step (1) above. Record the corresponding chromatograms. Calculate the content of cedrol in the test sample by peak area according to the external standard method. There should be no interfering peak at the retention time of the cedrol reference in the chromatogram of the negative sample solution.

9. The analytical detection method according to claim 8, characterized in that, In step (1), the chromatographic conditions are as follows: column: Fortis XI C8 (250×4.6 mm, 5 μm); mobile phase: acetonitrile-0.1% phosphoric acid aqueous solution (volume ratio 85:15); detection wavelength: 190 nm; column temperature: 30 ℃; flow rate: 1.0 mL / min; injection volume: 10 μL. In step (2), the cedrol reference solution is prepared as follows: accurately weigh 10 mg of cedrol reference standard, place it in a 50 mL volumetric flask, add a certain amount of acetonitrile to dissolve it by sonication, and dilute to the mark to obtain a 200 μg / mL cedrol reference solution. Seal and store at 4 ℃ for later use. In step (2), the preparation of the test solution is as follows: accurately pipette 1 mL of CE-NS suspension into a 50 mL volumetric flask, add acetonitrile, sonicate to dissolve and dilute to the mark to obtain the CE-NS test solution; In step (2), the negative sample solution is prepared by taking a negative sample that does not contain cedrol, which is composed of SDS, HPMC, PEG400 and water, and preparing it in the same way as the test sample solution.

10. The cedrol nano-suspension according to claim 1, characterized in that, The cedrol nanosuspension is used to prepare a drug that promotes hair regeneration in androgenetic alopecia.

Images