An amorphous assembly of oil-controlling, acne-removing, and antibacterial peptides, its preparation method, and its application.

Abstract

This invention belongs to the fields of biomedicine, beauty, and personal care health technology, and discloses an oil-controlling, acne-reducing, and antimicrobial peptide co-amorphous assembly, its preparation method, and applications. The method includes: mixing an octyl glycine solution with an antimicrobial peptide solution, rotary evaporation, and drying to obtain an antimicrobial peptide co-amorphous compound; mixing a cyclodextrin compound with water, adding the antimicrobial peptide co-amorphous compound, heating the reaction, and spray drying to obtain the oil-controlling, acne-reducing, and antimicrobial peptide co-amorphous assembly. The oil-controlling, acne-reducing, and antimicrobial peptide co-amorphous assembly provided by this invention efficiently solubilizes octyl glycine through co-amorphous technology and nano-assembly, improving its solubility and bioavailability; simultaneously optimizing the transdermal absorption of the antimicrobial peptide and achieving targeted accumulation in the dermis; the broad-spectrum antibacterial and oil-controlling effects of octyl glycine can be organically combined with the targeted antibacterial effects of the antimicrobial peptide, providing a new strategy for oily and acne-prone skin care; cyclodextrin inclusion enhances stability; the process is mild, simple, environmentally friendly, and easy to industrialize.

Description

Technical Field

[0001] This invention belongs to the fields of biomedicine, beauty and personal care health technology, and particularly relates to an amorphous assembly of oil-controlling, acne-removing and antibacterial peptides, its preparation method and application. Background Technology

[0002] With the growing severity of antibiotic resistance, the development of novel antimicrobial agents is urgently needed. Antimicrobial peptides, as a class of naturally derived small-molecule positively charged peptides, are considered important candidates to replace traditional antibiotics due to their multi-target mechanism of action and low likelihood of inducing resistance. However, their clinical application faces significant challenges: antimicrobial peptides have large molecular weights, making it difficult to penetrate the stratum corneum of the skin, resulting in low transdermal delivery efficiency; simultaneously, they are sensitive to proteases, have short half-lives in vivo, and are easily inactivated by environmental factors such as pH, oxidation, and hydrolysis, exhibiting poor physicochemical stability. To overcome these obstacles, local drug delivery combined with efficient delivery systems has become a key research direction.

[0003] Against this backdrop, caprylyl glycine, as a multifunctional amino acid derivative, has attracted widespread attention. Its molecular structure combines a lipophilic long caprylyl chain with a hydrophilic carboxylic acid amide group, endowing it with excellent amphiphilicity and skin affinity. Studies have shown that caprylyl glycine can effectively inhibit harmful skin bacteria such as Propionibacterium acnes and Staphylococcus aureus, and can regulate sebum secretion from the source by inhibiting 5α-reductase activity, achieving a dual effect of antibacterial and oil-controlling properties. More importantly, its amphiphilic structure theoretically allows it to interact with antimicrobial peptides. As a transdermal delivery carrier, it may not only improve the skin permeability and bioavailability of antimicrobial peptides, but its inherent broad-spectrum antibacterial properties can also provide synergistic preservative effects for formulations, reducing reliance on traditional chemical preservatives. However, caprylyl glycine itself is a lipid-soluble compound with extremely low solubility and poor water dispersibility, which severely limits its application in water-based formulations and its effectiveness as a carrier.

[0004] Currently, various technologies have been explored in the industry to improve the solubility and stability of capryloylglycine. Mainstream methods include encapsulation with liposomes to enhance its dispersibility in aqueous phase and transdermal absorption efficiency by forming nanoliposomes; or using composite emulsifiers (such as polyvinyl alcohol copolyphosphatidylcholine and carboxylated cholesterol) to construct stable oil-in-water emulsion systems, delaying release and increasing skin retention time. In addition, nanotechnology and microencapsulation techniques are also used to improve the stability of its active ingredients and achieve sustained-release efficacy. However, liposome encapsulation may suffer from unstable encapsulation rates and is prone to liposome rupture or drug leakage during long-term storage; while emulsion systems are more sensitive to formulation processes and storage conditions, and physical stability challenges remain.

[0005] Co-amorphous technology, by uniformly mixing poorly soluble drugs with excipients at the molecular level to form an amorphous solid, is more stable than the amorphous form of a single drug, significantly improving drug solubility and bioavailability. Combining capryloyl glycine with antimicrobial peptides through co-amorphous technology not only promises to improve the transdermal delivery efficiency of both but also integrates the targeted antibacterial activity of antimicrobial peptides with the broad-spectrum antibacterial and sebum-regulating effects of capryloyl glycine, providing a new strategy for multi-target treatment of acne. However, the amorphous form is thermodynamically metastable and tends to recrystallize spontaneously during storage. In particular, environmental moisture lowers the glass transition temperature of the system, acting as a plasticizer and accelerating the crystallization process, leading to the loss of its solubility advantage. Summary of the Invention

[0006] In order to solve the technical problems existing in the prior art, the purpose of this invention is to provide an oil-controlling, acne-removing, and antibacterial peptide co-amorphous assembly, its preparation method, and its application to solve the above-mentioned technical problems.

[0007] According to a first aspect of the present invention, the present invention provides a method for preparing an amorphous assembly of oil-controlling, acne-removing, and antibacterial peptides, comprising the following steps: (1) Dissolve octyl glycine in an organic solvent and heat to dissolve, to obtain an octyl glycine solution; dissolve the antimicrobial peptide in water and mix well to obtain an antimicrobial peptide solution; mix the octyl glycine solution and the antimicrobial peptide solution to obtain a mixture, rotary evaporate (remove solvent by rotary evaporation), and dry to obtain an antimicrobial peptide co-amorphous product; (2) Mix the cyclodextrin compound with water and stir until homogeneous to obtain a cyclodextrin compound solution. Add the antimicrobial peptide co-amorphous material. Heat the mixture under stirring to obtain an inclusion solution. Finally, spray dry the oil-controlling and acne-removing antimicrobial peptide co-amorphous assembly.

[0008] In some embodiments, the organic solvent in step (1) is at least one of methanol, ethanol, and isopropanol; the mass-to-volume ratio of the octanoylglycine to the organic solvent is 1:20-100 g / ml; and the heating and dissolving temperature is 40-50℃.

[0009] In some embodiments, the mass ratio of the antimicrobial peptide to capryloylglycine in step (1) is 1:3-15; In some embodiments, the amino acid sequence of the antimicrobial peptide in step (1) is shown in SEQ ID NO.1. Its amino acid sequence is NH2-Glu-Ser-Lys-Ile-Cys-Thr-Trp-Leu-Gly-Thr-Gly-Gln-Cys-Asn-Cys-Tyr-Lys-Cys-Lys-Trp-Glu-Gln-Ser-Leu-Gln-Cys-Arg-Asn-COOH.

[0010] The antimicrobial peptides selected in this invention have the following advantages: 1. Powerful and precise antibacterial effect: It has extremely low MIC values ​​(<0.50 μg / mL and <1.25 μg / mL) against key bacteria causing acne—Propionibacterium acnes and Staphylococcus aureus, respectively, with a high antibacterial rate. Its mechanism of action is to disrupt the bacterial cell membrane, rapidly kill bacteria, and is not prone to inducing drug resistance.

[0011] 2. Multi-effect: Not only does it remove acne, but it has also been experimentally proven to have anti-inflammatory, soothing, repairing and oil-controlling effects, which can improve acne problems from multiple targets.

[0012] 3. Highly safe and gentle: Safety assessments have confirmed that it is practically non-toxic and non-irritating. Suitable for all skin types, including sensitive skin.

[0013] In some embodiments, the mass-to-volume ratio of the antimicrobial peptide to water in step (1) is 1:100-1000 g / ml.

[0014] In some embodiments, the temperature of rotary evaporation in step (1) is 50-70°C; the temperature of drying is 40-60°C, and the drying time is 24-48h.

[0015] In some embodiments, the cyclodextrin compound in step (2) is at least one of β-cyclodextrin, hydroxypropyl-β-cyclodextrin, methyl-β-cyclodextrin, γ-cyclodextrin, and sulfobutyl ether-β-cyclodextrin; the mass-to-volume ratio of the cyclodextrin compound to water is (1-1.5):(40-60) g / mL; and the mass ratio of the cyclodextrin compound to the amorphous antimicrobial peptide is 1:0.5-5.

[0016] In some embodiments, the stirring speed in step (2) is 300-600 rpm; the temperature of the heating reaction is 40-60°C; and the heating reaction time is 2-6 h.

[0017] In some embodiments, the inlet temperature of the spray dryer in step (2) is 120-160°C, and the outlet temperature of the spray dryer is 60°C-80°C.

[0018] In some embodiments, the feed rate of the spray drying in step (2) is 0.2-0.6 L / h, and the atomizing gas flow rate is 2-8 m³ / h.

[0019] In some embodiments, the atomizing gas used for spray drying in step (2) is nitrogen.

[0020] According to a second aspect of the present invention, the present invention provides an amorphous assembly of oil-controlling, acne-removing, and antibacterial peptides prepared by the above-described preparation method.

[0021] According to a third aspect of the present invention, the present invention provides the application of an oil-controlling, acne-removing, and antibacterial peptide co-amorphous assembly in the preparation of topical pharmaceuticals or cosmetics.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) High efficiency solubilization and improved bioavailability: The oil-controlling, acne-removing, and antimicrobial peptide co-amorphous assembly constructed in this invention breaks the crystal structure of capryloylglycine without changing the molecular structure and performance of the drug, so that it exists in a high-energy disordered state and obtains a high drug loading, thereby significantly improving the apparent solubility and in vitro dissolution rate, providing an innovative solution to the core bottleneck of capryloylglycine application.

[0023] (2) Optimize transdermal absorption efficiency and bioavailability: This invention promotes the simultaneous delivery of antimicrobial peptides and capryloyl glycine through co-amorphization and nanoscale assembly. The transdermal amount is significantly higher than that of monomers, thereby improving the targeted accumulation in the dermis and enhancing efficacy.

[0024] (3) Synergistic effect, realizing full-pathway management of acne: The broad-spectrum antibacterial and oil-controlling effects of capryloyl glycine can be organically combined with the targeted antibacterial effect of antimicrobial peptides, providing a new strategy for the care of oily and acne-prone skin. The co-amorphous system enhances pharmacological activity, realizes skin care for oily skin acne removal, and makes its efficacy superior to that of a single component, achieving a therapeutic effect of 1+1>2.

[0025] (4) Excellent stability and strong formulation flexibility: The inclusion effect of the cyclodextrin selected in this invention provides a physical barrier for the co-amorphous assembly, effectively inhibiting water-induced plasticization and recrystallization, ensuring the long-term stability of the product, while further improving the drug solubility and dissolution performance, achieving multiple goals such as stability, solubilization, and controlled release.

[0026] (5) The preparation process is simple and easy to industrialize: the process conditions of this invention are mild, no complex equipment is required, the yield is high, it meets the requirements of green environmental protection, and it is easy to produce on a large scale. Attached Figure Description

[0027] Figure 1 A comparison diagram showing the solubility of the amorphous co-forms of octanoyl glycine in Test Example 1 and the antimicrobial peptides from Examples 1-3; Figure 2 The figure shows the solubility results of capryloyl glycine in the amorphous assemblies of the oil-controlling, acne-removing, and antibacterial peptides prepared in Examples 4-8 of Test Example 1 and the assemblies prepared in Comparative Examples 1-2. Figure 3 Polarized light microscope comparison images of capryloylglycine, antimicrobial peptide, physical mixture of the two monomers, and antimicrobial peptide co-amorphous product in test example 2; Figure 4The polarized light microscope images show the antimicrobial peptide co-amorphous compound, cyclodextrin, the physical mixture of the two, and the oil-controlling and acne-removing antimicrobial peptide co-amorphous assembly in Test Example 2. Figure 5 The dissolution curves of capryloyl glycine, the physical mixture of capryloyl glycine and antimicrobial peptide, the antimicrobial peptide co-amorphous product, and the oil-controlling and acne-removing antimicrobial peptide co-amorphous assembly were tested in Example 3. Figure 6 XRD comparison spectra of capryloyl glycine, antimicrobial peptide, antimicrobial peptide co-amorphous compound, and oil-controlling and acne-reducing antimicrobial peptide co-amorphous assembly in Example 3; Figure 7A DLS diagram of the amorphous assembly of the oil-controlling, acne-removing, and antibacterial peptides in Example 4; Figure 7B The Tyndall effect results of the co-amorphous assembly of the oil-controlling, acne-removing, and antibacterial peptides in Example 4 are shown in the figure. Figure 8 The transmission electron microscope images show the physical mixture and the amorphous assembly of the oil-controlling, acne-removing, and antibacterial peptides in Test Example 4. Figure 9 The images show a comparison of scanning electron microscopy (SEM) images of the physical mixture and the amorphous assembly of the oil-controlling, acne-reducing, and antimicrobial peptides in Test Example 4. Figure 10 Infrared comparison images of the physical mixture and the amorphous assembly of the oil-controlling, acne-reducing, and antibacterial peptides in Test Example 4; Figure 11 The diagram shows the interaction analysis between octanoyl glycine, the co-amorphous compound, and cyclodextrin in Test Example 5. Figure 12 ESP analysis plots of the antimicrobial peptide and co-amorphous compound in Test Example 5; Figure 13 Molecular frontier orbital analysis diagram of octanoylglycine and co-amorphous compound in test example 5; Figure 14 The figure shows the test results of the oil-controlling, acne-removing, and antibacterial peptide co-amorphous assembly of the present invention on the inhibition effect of 5α-reductase in Test Example 6; Figure 15 The figure shows the test results of the effect of the oil-controlling, acne-removing, and antibacterial peptide co-amorphous assembly of the present invention on the lipid droplet content of sebaceous gland cells in Test Example 6; Figure 16 The figure shows the experimental results of the antibacterial zone of the oil-controlling, acne-removing, and antimicrobial peptide co-amorphous assembly of the present invention in Test Example 7; Figure 17 Raman permeation comparison images of the oil-controlling, acne-removing, and antimicrobial peptide co-amorphous assembly, co-amorphous material, and antimicrobial peptide of the present invention in Test Example 8. Detailed Implementation

[0028] To better understand the technical solution of the present invention, the embodiments of the present invention will be described in detail below with reference to the accompanying drawings. It should be understood that the described embodiments are merely some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0029] Example 1 The preparation of the antimicrobial peptide co-amorphous compound includes the following steps: Weigh 3g of capryloylglycine and dissolve it in 150 mL of methanol. Heat at 40°C until completely dissolved to obtain an capryloylglycine solution. Separately weigh 1g of antimicrobial peptide and dissolve it in 100 mL of purified water. Mix well to obtain an antimicrobial peptide solution. Mix the two solutions, the capryloylglycine solution and the antimicrobial peptide solution, and stir at 50°C until clear and transparent to obtain a mixed solution. Then, remove the solvent by rotary evaporation under reduced pressure at 50°C. Place the obtained solid in a vacuum drying oven at 40°C for 48 hours to completely remove the residual solvent, thus obtaining the antimicrobial peptide amorphous product.

[0030] Example 2 The preparation of the antimicrobial peptide co-amorphous compound includes the following steps: Weigh 2.5g of capryloylglycine and dissolve it in 250 mL of isopropanol. Heat at 45°C until completely dissolved to obtain an capryloylglycine solution. Separately weigh 0.5g of antimicrobial peptide and dissolve it in 250 mL of purified water. Mix well to obtain an antimicrobial peptide solution. Mix the two solutions, the capryloylglycine solution and the antimicrobial peptide solution, and stir at 40°C until clear and transparent to obtain a mixed solution. Then, remove the solvent by rotary evaporation under reduced pressure at 60°C. Dry the obtained solid in a vacuum drying oven at 50°C for 36 hours to completely remove residual solvent, thus obtaining the antimicrobial peptide co-amorphous product.

[0031] Example 3 The preparation of the antimicrobial peptide co-amorphous compound includes the following steps: Weigh 6 g of capryloylglycine and dissolve it in 120 mL of ethanol. Heat at 50 °C until completely dissolved to obtain an capryloylglycine solution. Separately weigh 0.4 g of antimicrobial peptide and dissolve it in 400 mL of purified water. Mix well to obtain an antimicrobial peptide solution. Mix the above capryloylglycine solution and antimicrobial peptide solution and stir at 60 °C until clear and transparent to obtain a mixed solution. Then, remove the solvent by rotary evaporation under reduced pressure at 70 °C. Dry the obtained solid in a vacuum drying oven at 60 °C for 24 hours to completely remove residual solvent, thus obtaining the antimicrobial peptide co-amorphous product.

[0032] Example 4 The preparation method of the amorphous assembly of oil-controlling, acne-reducing, and antimicrobial peptides includes the following steps: (1) The preparation method of the antimicrobial peptide co-amorphous compound is the same as in Example 3.

[0033] (2) Weigh 1 g of cyclodextrin compound (β-cyclodextrin is selected), dissolve it in 60 mL of water, and stir magnetically at 300 rpm until completely dissolved. Add 5 g of the antimicrobial peptide co-amorphous material obtained in step (1), and set the stirring speed to 300 rpm. Heat the reaction under stirring (continuous stirring reaction at 40℃ for 6 h) to obtain a clear inclusion solution. Spray dry the inclusion solution. During the spray drying process, set the atomizing gas to nitrogen, the feed rate to 0.2 L / h, the atomizing gas flow rate to 3 m³ / h, the inlet temperature to 135℃, and the outlet temperature to 80℃. Collect the powder in the dryer to obtain the oil-controlling and acne-removing antimicrobial peptide co-amorphous assembly.

[0034] Example 5 The preparation method of the amorphous assembly of oil-controlling, acne-reducing, and antimicrobial peptides includes the following steps: (1) The preparation method of the antimicrobial peptide co-amorphous compound is the same as in Example 3.

[0035] (2) Weigh 1 g of cyclodextrin compound (hydroxypropyl-β-cyclodextrin is selected), dissolve it in 40 mL of water, and stir magnetically at 300 rpm until completely dissolved. Add 2.5 g of the antimicrobial peptide co-amorphous material obtained in step (1), and increase the stirring speed to 500 rpm. Heat the reaction under stirring (continuous stirring reaction at 60℃ for 2 h) to obtain a clear inclusion solution. Spray dry the inclusion solution. During the spray drying process, set the atomizing gas to nitrogen, the feed rate to 0.3 L / h, and the atomizing gas flow rate to 4 m³ / h. The inlet temperature is 120℃ and the outlet temperature is 60℃. Collect the powder in the dryer to obtain the oil-controlling and acne-removing antimicrobial peptide co-amorphous assembly.

[0036] Example 6 The preparation method of the amorphous assembly of oil-controlling, acne-reducing, and antimicrobial peptides includes the following steps: (1) The preparation method of the antimicrobial peptide co-amorphous compound is the same as in Example 3.

[0037] (2) Weigh 1.5 g of cyclodextrin compound (methyl-β-cyclodextrin is selected), dissolve it in 50 mL of water, and magnetically stir at 300 rpm until completely dissolved. Add 2.5 g of the antimicrobial peptide co-amorphous material obtained in step (1), and increase the stirring speed to 600 rpm. Heat the mixture under stirring (continuous stirring at 50℃ for 4 h) to obtain a clear inclusion solution. Spray dry the inclusion solution. During the spray drying process, set the atomizing gas to nitrogen, the feed rate to 0.6 L / h, and the atomizing gas flow rate to 2 m³ / h. The inlet temperature is 140℃ and the outlet temperature is 70℃. Collect the powder in a dryer to obtain the oil-controlling and acne-removing antimicrobial peptide co-amorphous assembly.

[0038] Example 7 The preparation method of the amorphous assembly of oil-controlling, acne-reducing, and antimicrobial peptides includes the following steps: (1) The preparation method of the antimicrobial peptide co-amorphous compound is the same as in Example 3.

[0039] (2) Weigh 2 g of cyclodextrin compound (sulfonyl butyl ether-β-cyclodextrin is selected), dissolve it in 55 mL of water, and magnetically stir at 300 rpm until completely dissolved. Add 2.5 g of the antimicrobial peptide co-amorphous material obtained in step (1), and increase the stirring speed to 400 rpm. Heat the mixture under stirring (continuous stirring at 55℃ for 2.5 h) to obtain a clear inclusion solution. After the inclusion solution reaction is completed, spray dry it. During the spray drying process, set the atomizing gas to nitrogen, the feed rate to 0.4 L / h, and the atomizing gas flow rate to 8 m³ / h. The inlet temperature is 160℃ and the outlet temperature is 65℃. Collect the powder in a dryer to obtain the oil-controlling and acne-removing antimicrobial peptide co-amorphous assembly.

[0040] Example 8 The preparation method of the amorphous assembly of oil-controlling, acne-reducing, and antimicrobial peptides includes the following steps: (1) The preparation method of the antimicrobial peptide co-amorphous compound is the same as in Example 3.

[0041] (2) Weigh 4 g of a cyclodextrin compound (γ-cyclodextrin is selected), dissolve it in 45 mL of water, and magnetically stir at 300 rpm until completely dissolved. Add 2 g of the antimicrobial peptide co-amorphous material obtained in step (1), and increase the stirring speed to 450 rpm. Heat the mixture under stirring (continuous stirring at 45℃ for 5 h) to obtain a clear inclusion solution. Spray dry the inclusion solution. During the spray drying process, set the atomizing gas to nitrogen, the feed rate to 0.5 L / h, and the atomizing gas flow rate to 5 m³ / h. The inlet temperature is 150℃, the outlet temperature is 75℃, and collect the powder in a dryer to obtain the oil-controlling and acne-removing antimicrobial peptide co-amorphous assembly.

[0042] Comparative Example 1 To investigate the inclusion effect of cyclodextrin on capryloylglycine raw material, Comparative Example 1 provides a method for preparing an assembly. This method is basically the same as that in Example 5, except that the antimicrobial peptide co-amorphous material added in Comparative Example 1 is changed to capryloylglycine raw material, and other experimental operations are the same as in Example 5.

[0043] Comparative Example 2 To investigate the inclusion effect of cyclodextrin on the physical mixture of capryloylglycine and antimicrobial peptides, Comparative Example 2 provides a method for preparing an assembly that is basically the same as that in Example 5, except that the antimicrobial peptide co-amorphous material added in Comparative Example 2 is adjusted to a physical mixture of capryloylglycine and antimicrobial peptides, and other experimental operations are the same as in Example 5.

[0044] The preparation of the physical mixture of the capryloyl glycine raw material and the antimicrobial peptide raw material includes: mixing capryloyl glycine and the antimicrobial peptide, stirring evenly to obtain the physical mixture of the capryloyl glycine raw material and the antimicrobial peptide raw material; the mass ratio of capryloyl glycine to antimicrobial peptide is 15:1.

[0045] Test Example 1: Solubility Test of Antimicrobial Peptide Co-amorphous Compound Solubility Test 1: 10 g of the products from Examples 1-3 (antimicrobial peptide co-amorphous products) and capryloylglycine raw material were added to 10 mL of pure water, respectively. After equilibration at 25°C and 300 rpm for 24 h, the mixture was filtered, and the equilibrium solubility of capryloylglycine was determined by HPLC. Three parallel experiments were performed, and the results are as follows: Figure 1 As shown, the formation of the antimicrobial peptide co-amorphous compound significantly improves the solubility of capryloylglycine, with the increase in solubility increasing with the increase in antimicrobial peptide content. Considering both the amount of antimicrobial peptide added and the effect on improving the solubility of capryloylglycine, the process of Example 3 was selected for further research, and its solubility was increased by 5.78 times compared to the capryloylglycine raw material.

[0046] Solubility Test 2: 10 g of the products from Examples 4-8 (amorphous assemblies of oil-controlling, acne-reducing, and antibacterial peptides) were added to 10 mL of pure water. After equilibration at 25°C and 300 rpm for 24 h, the mixture was filtered, and the equilibrium solubility of capryloyl glycine was determined by HPLC. The experiment was performed in triplicate, and the results are as follows: Figure 2As shown, the inclusion effect of cyclodextrin further improves the solubility of capryloylglycine, but the effect of different types of cyclodextrin on the solubility improvement of capryloylglycine varies significantly. The solubility of capryloylglycine in the products of Examples 4-8 is 64.22, 115.61, 102.76, 89.92, and 54.59 times that of the active pharmaceutical ingredient, respectively. Therefore, Example 5 was selected as the research object for subsequent studies. The solubility of capryloylglycine in the products of Comparative Examples 1 and 2 is significantly lower than that in Example 5, indicating that the inclusion effect of cyclodextrin on the antimicrobial peptide co-amorphous material is better than that on the capryloylglycine active pharmaceutical ingredient and its physical mixture with the antimicrobial peptide. This result may be due to the extremely high instantaneous solubility of the co-amorphous material, which allows more capryloylglycine to smoothly enter the cavities of the cyclodextrin.

[0047] Test Example 2: Comparison of Polarized Microscopy Results of Antimicrobial Peptide Co-amorphous Compounds Experiment 1: The amorphous product of capryloyl glycine, antimicrobial peptide, a physical mixture of the two (capryloyl glycine and antimicrobial peptide mixed at a mass ratio of 15:1 and stirred until homogeneous), and the antimicrobial peptide prepared in Example 3 were observed under a polarizing microscope. Results are as follows: Figure 3 As shown.

[0048] Figure 3 Polarized light microscope images of the antimicrobial peptide active pharmaceutical ingredient, capryloyl glycine active pharmaceutical ingredient, their physical mixture, and the antimicrobial peptide co-amorphous product (the product of Example 3) are shown. Figure 3 It can be seen that the antimicrobial peptide raw material, capryloyl glycine raw material, and their physical mixture all exhibit typical crystalline characteristics, with regular geometric shapes, sharp grain boundaries, and obvious birefringence. In contrast, the antimicrobial peptide co-amorphous compound produced in Example 3 is amorphous, exhibiting a uniformly dark field of view and no birefringence under crossed polarized light, indicating that the antimicrobial peptide and capryloyl glycine have been successfully prepared as a co-amorphous compound.

[0049] Experiment 2: The amorphous assembly of antimicrobial peptides (product of Example 3), cyclodextrin compounds (hydroxypropyl-β-cyclodextrin was selected), a physical mixture of the three (hydroxypropyl-β-cyclodextrin, capryloyl glycine, and antimicrobial peptides were mixed in a mass ratio of 6:15:1 and stirred until homogeneous), and the oil-controlling and acne-reducing antimicrobial peptides prepared in Example 5 were observed under a polarizing microscope. The results are as follows: Figure 4 As shown.

[0050] Figure 4Polarized light micrographs of the amorphous antimicrobial peptide, cyclodextrin, a physical mixture of the three, and the amorphous assembly of the oil-controlling and acne-reducing antimicrobial peptide are presented. It can be seen that although cyclodextrin exhibits a certain dark color, it remains a well-defined, regularly shaped spherical or ellipsoidal semi-crystalline substance. Physical mixing did not alter the semi-crystalline state of the cyclodextrin. After forming the amorphous assembly of the oil-controlling and acne-reducing antimicrobial peptide, the overall structure is a highly diffused dark black region with significantly refined particle size, indicating that cyclodextrin achieved molecular-level inclusion of the amorphous antimicrobial peptide, forming a new substance, namely the amorphous assembly of the oil-controlling and acne-reducing antimicrobial peptide.

[0051] Test Example 3: Dissolution performance test and crystal structure observation of antimicrobial peptide co-amorphous compounds and oil-controlling and acne-reducing antimicrobial peptide co-amorphous assemblies. The amorphous antimicrobial peptide compound (product of Example 3), the amorphous assembly of oil-controlling and acne-reducing antimicrobial peptide compound (product of Example 5), capryloyl glycine raw material, and antimicrobial peptide raw material were ground and then passed through 100-mesh and 200-mesh sieves sequentially to control the powder particle size between 75 and 150 μm, avoiding dissolution differences caused by differences in powder particle size. 100 g of the ground amorphous antimicrobial peptide compound (product of Example 3), the amorphous assembly of oil-controlling and acne-reducing antimicrobial peptide compound (product of Example 5), the capryloyl glycine raw material, and a physical mixture of the capryloyl glycine raw material and the antimicrobial peptide raw material (the two raw materials were ground, sieved, and mixed at a mass ratio of 15:1, and stirred until homogeneous) were added to 100 mL of pure water at a time. The mixture was kept at a constant temperature of 25°C and continuously stirred at a rate of 300 rpm. 1 mL samples were taken at 10 min, 20 min, 30 min, 1 h, and 2 h after the addition of the drugs. After filtration, the concentration of capryloyl glycine was determined by HPLC. The experiment was conducted in triplicate. The concentration of octanoyl glycine in the solution was calculated at each time point, and the dissolution curves for each sample were plotted. The results are as follows: Figure 5 As shown. Figure 5 The term "amorphous assembly" refers to the product of Example 5, "co-amorphous material" refers to the product of Example 3, "physical mixture" refers to the physical mixture of capryloylglycine raw material and antimicrobial peptide raw material, and "capryloylglycine" refers to capryloylglycine raw material.

[0052] like Figure 5As shown, different forms of caprylyl glycine exhibited significantly different release behaviors in the dissolution experiment. The caprylyl glycine active pharmaceutical ingredient (API) itself had poor solubility; its concentration slowly increased over 30 minutes, stabilizing near saturation. The dissolution curve of the physical mixture was similar to that of the API, also approaching saturation after 30 minutes, but the solubility of caprylyl glycine was somewhat improved. In the antimicrobial peptide co-amorphous assembly, the dissolution behavior of caprylyl glycine exhibited a typical "spring-parachute" effect: the concentration rapidly reached its maximum value within a short time, then decreased rapidly, consistent with its high-energy state characteristics. In contrast, in the oil-controlling and acne-reducing antimicrobial peptide co-amorphous assembly, caprylyl glycine dissolved rapidly and remained stable throughout the test after approaching saturation, indicating that this system possesses the dual advantages of rapid drug release and long-term stability. This demonstrates that the cyclodextrin inclusion technology not only further enhances the solubility of caprylyl glycine but also effectively improves the instability problem of the co-amorphous system.

[0053] XRD testing: The crystal structure of the antimicrobial peptide, capryloyl glycine, the amorphous antimicrobial peptide (product of Example 3), and the amorphous assembly of the oil-controlling and acne-reducing antimicrobial peptide (product of Example 5) was tested by XRD. The results are as follows: Figure 6 As shown.

[0054] As can be seen, both capryloylglycine and antimicrobial peptides exhibit numerous and sharp diffraction peaks, a typical characteristic of highly crystalline substances. In the amorphous antimicrobial peptide co-assembly, the sharp diffraction peaks almost completely disappear. This indicates that the formation of the amorphous antimicrobial peptide co-assembly greatly inhibits crystallization. Meanwhile, cyclodextrin is mainly in an amorphous form with a small number of crystalline regions. Compared to cyclodextrin, the diffraction curve of the amorphous assembly of the oil-controlling and acne-reducing antimicrobial peptide co-assembly is smoother, with no obvious diffraction peaks. This indicates that while cyclodextrin exerts its inclusion effect, it also forms a more complex and stable ternary amorphous complex system.

[0055] Test Example 4: Microstructural Characterization of Amorphous Assemblies of Oil-Controlling, Acne-Clearing, and Antibacterial Peptides Tyndall effect and particle size distribution: Six g of the oil-controlling, acne-reducing, and antimicrobial peptide co-amorphous assembly (the product of Example 5) was dissolved in 100 mL of deionized water and stirred at 25°C and 300 rpm for 30 min, followed by ultrasonic treatment for 10 min to ensure thorough dispersion. Subsequently, its Tyndall effect and particle size distribution were tested, and the results are as follows: Figure 7A and Figure 7B As shown. The amorphous assembly solution of the oil-controlling, acne-reducing, and antibacterial peptides exhibits a significant Tyndall effect, indicating that it exists in the form of nanoparticles in solution. The particle size distribution results show ( Figure 7AThe average particle size is about 224 nm, and the polydispersity index (PDI) is as low as 0.0651, indicating that the particle size distribution of the system is highly uniform and almost monodisperse.

[0056] Transmission electron microscopy (TEM) observation: The amorphous assembly of the oil-controlling, acne-reducing, and antibacterial peptides (product of Example 5) and a physically mixed sample (physical mixture prepared in Comparative Example 2) were observed under a transmission electron microscope. The TEM results are as follows: Figure 8 As shown. Figure 8 The left side of the sample is a physically mixed sample. Sparse, coarse and irregularly shaped active pharmaceutical ingredient (API) particles are visible in the field of view, indicating that physical mixing did not change the poor water solubility of the API. Figure 8 The right side of the structure is an amorphous assembly of oil-controlling, acne-removing, and antibacterial peptides, exhibiting densely and uniformly distributed nanoscale particles with uniform size and near-spherical morphology. This structure provides direct morphological evidence for the molecular-level assembly of components and the formation of new substances.

[0057] Scanning electron microscopy (SEM) observation: To facilitate storage and transportation, the amorphous assembly of the oil-controlling, acne-reducing, and antimicrobial peptides (product of Example 5) and the physically mixed sample (physical mixture prepared in Comparative Example 2) were dried and pulverized separately before being observed and analyzed under a scanning electron microscope. The SEM results are as follows: Figure 9 As shown. Figure 9 The sample on the left is a physically mixed sample. Figure 9 The right side shows an amorphous assembly of oil-controlling, acne-reducing, and antibacterial peptides. (Example:) Figure 9 As shown, the physically mixed sample clearly exhibits two different morphologies, displaying a typical heterogeneous structure. In contrast, the co-amorphous assembly after drying and pulverization displays a single, continuous amorphous solid morphology with smooth edges. This morphology confirms that the two molecules have formed a uniform, single new phase at the molecular level, indicating that the drying and pulverization process did not destroy its amorphous structure or affect the inclusion state of the cyclodextrin.

[0058] Fourier Transform Infrared Spectroscopy Analysis: The amorphous assembly of the oil-controlling, acne-reducing, and antibacterial peptides (product of Example 5) and the physically mixed sample (physical mixture prepared in Comparative Example 2) were respectively tested using a Fourier Transform Infrared Spectrometer (FTIR). The results are as follows: Figure 10 As shown. Figure 10 The amorphous assembly of the oil-controlling, acne-reducing, and antimicrobial peptides in Example 5 represents the product of Example 5, while the physical mixture represents the physical mixture prepared in Comparative Example 2. The spectrum of the physically mixed sample shows a series of sharp, separated characteristic absorption peaks, corresponding to the independent functional group vibrations of each component, indicating that the two only physically coexist without significant intermolecular interactions. In contrast, the absorption peaks of the amorphous assembly of the oil-controlling, acne-reducing, and antimicrobial peptides are significantly broadened, with some peaks shifting or even merging, especially in the vibrational regions of hydroxyl, amino, and characteristic carbonyl groups (e.g., 3000-3500 cm⁻¹).-1 With 1600-1700 cm -1 The effect is even more pronounced in the vicinity. This phenomenon indicates that a homogeneous amorphous solid system is formed between the components through strong intermolecular forces, the vibrations of the original crystalline phase are suppressed, and the spectral bands tend to be single and continuous, thus confirming the formation of a co-amorphous phase with new interactions at the molecular level.

[0059] Test Example 5: Simulated Test of Oil-Controlling, Acne-Reducing, and Antimicrobial Peptide Co-Amorphous Assemblies and Antimicrobial Peptide Co-Amorphous Compounds To investigate the molecular interaction mechanism in the amorphous assembly of the oil-controlling, acne-reducing, and antibacterial peptides, density functional theory (DFT) was used in Gaussian software for computational analysis. The molecular interactions between the amorphous components and cyclodextrin in the amorphous assembly of the oil-controlling, acne-reducing, and antibacterial peptides of this invention (product of Example 5) were analyzed. Simultaneously, the molecular interactions between capryloyl glycine and cyclodextrin in the product of Comparative Example 1 were analyzed using Gaussian software. The results are as follows: Figure 11 As shown. Due to the large molecular weight of the antimicrobial peptide, it could not be simulated with its complete structure. Based on the working principle of the antimicrobial peptide, a positively charged peptide fragment, Tyr-Lys-Cys-Lys-Trp, was used for simulation calculations. This combination contains two positively charged Lys, a strongly hydrophobic Trp with a large π-electron system (crucial for disrupting membrane structure), and an aromatic hydrophobic Tyr, exhibiting excellent charge-hydrophobic balance and making it an ideal candidate for simulating the antimicrobial peptide. Independent gradient model (IGMH) based on Hirshfeld segmentation and atomic in-molecule (AIM) topological analysis showed that the binding energy between the antimicrobial peptide co-amorphous compound and cyclodextrin was -118.24 kcal / mol, significantly better than the binding energy between the monomer and cyclodextrin (-66.13 kcal / mol). This indicates a stronger intermolecular interaction between the co-amorphous compound and cyclodextrin, resulting in an inclusion complex with higher thermodynamic stability.

[0060] Figure 12 This is a surface electrostatic potential (ESP) distribution diagram of the amorphous compound of capryloylglycine and antimicrobial peptide (product of Example 3). The positive and negative potentials of capryloylglycine are mainly concentrated in the head, consistent with its amphiphilic characteristics. Due to the folding of the polypeptide chain, the antimicrobial peptide exhibits significant spatial heterogeneity in its ESP distribution, with locally concentrated negative potential regions and dispersed positive potential regions. The amorphous compound of the two antimicrobial peptides shows different ESP characteristics: the original charge boundaries are broken, and the positive and negative potential regions interpenetrate and tend to become homogeneous. The charge rearrangement and delocalization phenomena indicate that there are strong electrostatic interactions and electron cloud overlap between molecules in the amorphous system, confirming its enhanced intermolecular forces and higher structural homogeneity at the electronic structure level.

[0061] Figure 13The molecular frontier orbital analysis results of the antimicrobial peptide and its co-amorphous form (product of Example 3) are presented. Based on the energy and spatial distribution analysis of the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO), the band gap (ΔE) of capryloylglycine is 4.80 eV, and that of the antimicrobial peptide is 3.46 eV, indicating that both require relatively high energies for electronic excitation. The ΔE of the co-amorphous form is significantly reduced to 3.34 eV, lower than that of any monomer, indicating a decreased energy barrier for electron transitions from the HOMO to the LUMO. This suggests a potential enhancement in reactivity or charge transfer efficiency, revealing a potential mechanism for changes in the physicochemical behavior of the system at the electronic structure level.

[0062] Test Example 6: Oil-controlling, acne-reducing, and antibacterial peptide co-amorphous assembly oil-controlling efficacy test 5α-Reductase Inhibition Effect Test: Amorphous assemblies of oil-controlling, acne-reducing, and antimicrobial peptides and capryloylglycine were used as samples. The oil-controlling efficacy of the samples was evaluated according to the "Biochemical Experimental Method for 5α-Reductase Inhibition Rate". The reagents used were as follows: 5α-reductase (provided by Suzhou Huizhiheyuan Biotechnology Co., Ltd.), dutasteride (provided by Shanghai Maclean Biochemical Technology Co., Ltd.), testosterone (provided by Beijing Wokai Biotechnology Co., Ltd.), and tetrasodium reductase II (NADPH, provided by Shanghai Maclean Biochemical Technology Co., Ltd.). The experimental procedures are described below.

[0063] Referring to Table 1, sample tubes (T), sample background (T0), blank tubes (C), and blank background (C0) were set up. Each sample at each test concentration in sample tubes (T) required three parallel measurements, and the blank tubes (C) also required three parallel measurements. First, 440 μL of PBS buffer was added to all test tubes. Then, 40 μL of the same concentration of sample solution was added to each of the sample wells (T) and sample background (T0), while 40 μL of PBS buffer was added to the blank tubes (C) and blank background (C0). Next, 40 μL of testosterone solution, 120 μL of reduced coenzyme II solution, and 160 μL of 5α-reductase solution were added to the sample tubes (T) and blank tubes (C), while 320 μL of PBS buffer was added to the sample background (T0) and blank background (C0). The absorbance was measured at 340 nm after sample addition. The absorbance values ​​at this point were recorded as follows: Asample = A0min (sample 0 min) and Ablank = A0min (blank matrix 0 min). Subsequently, the sample was incubated at 37°C for 60 min, and the absorbance was measured again at 340 nm. The absorbance values ​​of the sample after subtracting the sample background (Asample60 min) and the absorbance values ​​of the blank matrix after subtracting the blank background (Ablank60 min) were recorded.

[0064] Table 1 Sample Addition Requirements Each sample was tested in triplicate, and the inhibition rate of 5α-reductase activity was calculated using the following formula: Inhibition rate (%) = [(A 空0min -A 空60min )-(A 样0min -A 样60min )] / (A empty 0min -A 空60min ) 100% In the formula: A 空0min —The average of three absorbance values ​​obtained from the blank tube at 0 min after subtracting the blank background; A 空60min —The average of three absorbance values ​​obtained from the blank tube after subtracting the blank background, measured over 60 minutes; A 样0min —The absorbance value of the sample tube obtained from the 0-minute measurement, after deducting the sample background, needs to be measured in parallel 3 times; A 样60min —The absorbance value of the sample tube obtained after 60 minutes of measurement, after deducting the sample background, needs to be measured in parallel 3 times.

[0065] The inhibition rate of 5α-reductase activity at a given test concentration is expressed as mean inhibition rate ± standard deviation (SD). P < 0.05 was considered statistically significant, and P < 0.01 was considered highly statistically significant.

[0066] The sample groups included capryloyl glycine group 1, capryloyl glycine group 2, co-amorphous assembly group 1, and co-amorphous assembly group 2. Specifically, in capryloyl glycine group 1, the concentration of capryloyl glycine in the sample solution was 0.09 wt%; in capryloyl glycine group 2, the concentration of capryloyl glycine in the sample solution was 0.15 wt%; in co-amorphous assembly group 1, the concentration of capryloyl glycine released from the oil-controlling, acne-reducing, and antimicrobial peptide co-amorphous assembly in the solution was 0.09 wt%; and in co-amorphous assembly group 2, the concentration of capryloyl glycine released from the oil-controlling, acne-reducing, and antimicrobial peptide co-amorphous assembly in the solution was 0.15 wt%. Figure 14 This study demonstrates a comparison of the inhibitory effects of the oil-controlling, acne-reducing, and antimicrobial peptide co-amorphous assembly and capryloylglycine on 5α-reductase activity at different concentrations. Figure 14In this context, "caprylyl glycine 1" represents the test result of caprylyl glycine at a concentration of 0.09 wt%, "co-amorphous assembly 1" represents the test result of the oil-controlling, acne-reducing, and antimicrobial peptide co-amorphous assembly released in solution at a concentration of 0.09 wt%, "caprylyl glycine 2" represents the test result of caprylyl glycine at a concentration of 0.15 wt%, and "co-amorphous assembly 2" represents the test result of the oil-controlling, acne-reducing, and antimicrobial peptide co-amorphous assembly released in solution at a concentration of 0.15 wt%. The preparation method of the oil-controlling, acne-reducing, and antimicrobial peptide co-amorphous assembly can be referred to Example 5.

[0067] The results show that ( Figure 14 At a capryloyl glycine concentration of 0.09 wt%, the enzyme activity inhibition rate of the amorphous assembly of the oil-controlling and acne-reducing antimicrobial peptide was increased by 21.4% compared with the same concentration of capryloyl glycine monomer. When the concentration increased to 0.15%, the inhibition rate was further significantly enhanced and was significantly better than that of the same concentration of capryloyl glycine group, indicating that its inhibitory effect was dose-dependent. This result demonstrates that, compared with capryloyl glycine monomer, the amorphous assembly of the oil-controlling and acne-reducing antimicrobial peptide has better oil-controlling potential at equivalent concentrations.

[0068] Sebaceous gland cell lipid droplet content test: The oil-control efficacy of the sample was evaluated according to the "Standard Operating Procedure for Determination of Lipid Droplet Content in In Vitro Human Sebaceous Gland Cells in Cosmetic Oil Control Efficacy Testing". The sample consisted of an amorphous assembly of oil-controlling, acne-reducing, and antibacterial peptides prepared in Example 5, along with capryloyl glycine. The principle is as follows: Sebaceous glands are the source and target tissue of androgens. Dehydroepiandrosterone (DHEA) is produced in sebaceous glands, which is converted into the most active testosterone and 5α-dihydrotestosterone through a series of enzymatic catalysis. These stimulate lipid synthesis and the proliferation and differentiation of sebaceous gland cells. During this proliferation and differentiation process, undifferentiated immature cells proliferate first, and some daughter cells form lipid droplets, which migrate towards the center of the gland cell and gradually develop and differentiate into mature sebaceous gland cells capable of storing and secreting lipids. This experiment used Nile Red staining to detect the amount of oil secreted by sebaceous gland cells, thereby determining whether the sample has oil-controlling efficacy. The sebaceous gland cells used were purchased from Hangzhou Ruixu Kexin Biotechnology Co., Ltd.; the culture medium used was DMEM medium, purchased from Thermo Fisher Scientific (China) Co., Ltd., catalog number C11995500BT.

[0069] 1. Cells were seeded in 12-well plates and incubated at 37°C with 5% CO2 for 24 hours. The experimental groups are shown in Table 2 below.

[0070] Table 2 2. After incubation, remove the culture medium and wash 1-2 times with D-Hanks buffer. Replace the normal control group with fresh culture medium, the model control group with fresh culture medium containing dihydrotestosterone, and the sample group with fresh culture medium containing the sample and dihydrotestosterone. Perform 3 biological replicates and continue incubation at 37°C and 5% CO2 for 24 hours.

[0071] 3. After incubation, remove the culture medium, pre-cool to 4°C, wash three times with D-Hanks buffer, fix with 4% paraformaldehyde, wash with D-Hanks buffer, stain with Nile Red, take pictures under a fluorescence microscope, and analyze the average fluorescence intensity of each group using ImageJ.

[0072] 4. The oil-controlling effect was calculated using the following formula. P<0.05 was considered statistically significant, and P<0.01 was considered highly statistically significant.

[0073] Oil control efficacy (%) = (A 模型对照组 -A 样品组 ) / A 模型对照组 100% Where: A — average fluorescence intensity The sample groups in Table 2 include an isoconcentration caprylyl glycine group and a co-amorphous assembly group; the drug administration concentration (the concentration of caprylyl glycine released from the oil-controlling and acne-reducing antimicrobial peptide co-amorphous assembly in the culture medium) in the co-amorphous assembly group is 0.03 wt%, and the corresponding concentration of the oil-controlling and acne-reducing antimicrobial peptide co-amorphous assembly in the culture medium is 0.1 wt%; while the drug administration concentration (the concentration of caprylyl glycine in the culture medium) in the isoconcentration caprylyl glycine group is 0.03 wt%.

[0074] Figure 15 The fluorescence intensity of the amorphous assembly of the oil-controlling and acne-reducing antimicrobial peptide and capryloyl glycine on the lipid droplet content of sebaceous gland cells was measured. The average fluorescence intensity of the model control group was significantly higher than that of the normal control group, indicating that the stimulation condition was effective. When the concentration of capryloyl glycine released from the amorphous assembly of the oil-controlling and acne-reducing antimicrobial peptide in the culture medium was 0.03 wt%, the fluorescence intensity of the amorphous assembly group was significantly lower than that of the model control group by 30%, indicating that the amorphous assembly has good oil-controlling efficacy. Meanwhile, there was no significant difference between the test results of the amorphous assembly and those of capryloyl glycine, meaning that the amorphous technology did not adversely affect the oil-controlling effect of capryloyl glycine.

[0075] Test Example 7: Acne-removing efficacy test of oil-controlling, acne-removing, and antibacterial peptide co-amorphous assemblies. The occurrence of acne is closely related to the abnormal proliferation of Propionibacterium acnes. Excessive proliferation of this bacterium within hair follicles can decompose sebum and induce local inflammatory responses, promoting the release of inflammatory factors such as IL-6, TNF-α, and IL-8, thereby exacerbating redness, swelling, and inflammatory infiltration. Using Propionibacterium acnes (purchased from the China Industrial Microbial Culture Collection Center, catalog number 0170L) as a model, an inhibition zone experiment was conducted to assess the acne-reducing potential of the sample: the larger the diameter of the inhibition zone, the stronger its inhibitory ability on bacterial proliferation, and the more significant the acne-reducing effect. The specific experimental steps are as follows. The culture medium used was Propionibacterium acnes solid culture medium (purchased from Shanghai Ruichu Biotechnology Co., Ltd., catalog number T2283); the positive control used was kanamycin (purchased from Shanghai Beyotime Biotechnology Co., Ltd., catalog number ST101).

[0076] The inhibition zone test includes the following steps: 1. After bacterial resuscitation, passage twice to expand the culture.

[0077] 2. Autoclave the culture medium in advance and cool it to 60°C (to avoid burns and prevent solidification).

[0078] 3. In a sterile operating table, pour the sterilized culture medium into sterile petri dishes, about 5 ml per dish, avoiding the formation of air bubbles.

[0079] 4. Shake the petri dish to spread the culture medium evenly, and let it stand on a horizontal surface to solidify naturally (30 minutes). 5. After solidification, invert the petri dish and place it in an anaerobic culture device for pre-culture for 24 hours. Once confirmed to be free of contamination, it can be used for inoculation of Propionibacterium acnes to ensure a sterile environment for the inhibition zone detection experiment.

[0080] 6. Add 200 μL of bacterial suspension to each plate (bacterial density in the suspension is 1.5). 10 8 (CFU / mL), spread evenly with a coating stick, add uniformly sized circular filter paper, and drop the liquid to be tested onto it.

[0081] 7. Experimental groups: The experiment was divided into a negative control group (NC), a positive control group (PC), and a sample group, as shown in Table 3 below.

[0082] Table 3 8. Invert the petri dish and place it in the anaerobic culture equipment for pre-culture for 72 hours; 9. Take photos at the same height using a camera, and use ImageJ to calculate the diameter of the inhibition zone.

[0083] The sample groups in Table 3 include the co-amorphous assembly group, the capryloyl glycine group, and the antimicrobial peptide group. In the co-amorphous assembly group, the concentration of the oil-controlling and acne-reducing antimicrobial peptide co-amorphous assembly in the test liquid is 0.1 wt%, the concentration of capryloyl glycine released into the test liquid is 0.03 wt%, and the concentration of antimicrobial peptide released into the test liquid is 0.002 wt%. In the capryloyl glycine group, the concentration of capryloyl glycine in the test liquid is 0.03 wt%, and in the antimicrobial peptide group, the concentration of antimicrobial peptide in the test liquid is 0.002 wt%.

[0084] Figure 16 This study demonstrated the antibacterial effects of an amorphous assembly of an oil-controlling and acne-reducing antimicrobial peptide, capryloyl glycine, and the antimicrobial peptide against *Propionibacterium acnes*. Compared to the negative control group, the diameter of the inhibition zone in the positive control group was significantly increased, indicating that the experimental system was normal. When the amorphous assembly of the oil-controlling and acne-reducing antimicrobial peptide was released into the test liquid at a concentration of 0.1 wt% of the antimicrobial peptide, the diameter of the inhibition zone of the amorphous assembly was 29.82% larger than that of the negative control group, exhibiting significant antibacterial activity. At the same concentration, the diameters of the inhibition zones of capryloyl glycine and the antimicrobial peptide increased by only 6.10% and 13.97%, respectively. These results indicate that the amorphous assembly of the oil-controlling and acne-reducing antimicrobial peptide not only improves the solubility and stability of the drug but also significantly enhances its antibacterial efficacy through intermolecular synergistic effects, achieving a pharmacological synergistic effect of "1+1>2".

[0085] Test Example 8: Transdermal Absorption Experiment of Oil-Controlling, Acne-Clearing, and Antibacterial Peptide Co-Amorphous Assemblies Referring to the group standard T / ZGKSL010-2023 "Method for Determination of Transdermal Absorption of Recombinant Collagen", FITC-labeled antimicrobial peptides were used. Then, following the method described in Example 3, FITC-labeled antimicrobial peptides were used as raw materials to prepare an amorphous assembly of antimicrobial peptides for oil control and acne treatment. The transdermal absorption capacity of the antimicrobial peptides in pig skin was then evaluated. The fluorescent labeling results are shown below. Figure 17 As shown in the figure. The results showed that after forming the co-amorphous compound, the cumulative skin penetration of the antimicrobial peptide increased by 127%, indicating that the co-amorphous technology can significantly improve its transdermal performance; after further forming the co-amorphous assembly, the penetration increased by 368%, which confirms that cyclodextrin inclusion has a significant permeation-enhancing effect on the co-amorphous compound, which may be due to the improved stability of the co-amorphous compound in the nano-assembly.

[0086] The above descriptions are merely some embodiments of the present invention. Those skilled in the art can make various modifications and improvements without departing from the inventive concept of the present invention, and these all fall within the scope of protection of the present invention.

Claims

1. A method for preparing an amorphous assembly of oil-controlling, acne-removing, and antibacterial peptides, characterized in that, Includes the following steps: (1) Dissolve octyl glycine in an organic solvent and heat to dissolve, to obtain an octyl glycine solution; dissolve the antimicrobial peptide in water to obtain an antimicrobial peptide solution; mix the octyl glycine solution and the antimicrobial peptide solution to obtain a mixture, evaporate by rotary evaporation and dry to obtain an antimicrobial peptide amorphous product; (2) Mix the cyclodextrin compound with water to obtain a cyclodextrin compound solution, add the antimicrobial peptide co-amorphous material, heat the mixture under stirring to obtain an inclusion solution, spray dry to obtain the oil-controlling and acne-removing antimicrobial peptide co-amorphous assembly.

2. The method for preparing the oil-controlling, acne-removing, and antibacterial peptide co-amorphous assembly according to claim 1, characterized in that, The organic solvent in step (1) is at least one of methanol, ethanol, and isopropanol; the mass-volume ratio of the octanoylglycine to the organic solvent is 1:20-100 g / ml; and the heating and dissolving temperature is 40-50℃.

3. The method for preparing the oil-controlling, acne-removing, and antibacterial peptide co-amorphous assembly according to claim 1, characterized in that, The mass ratio of the antimicrobial peptide to capryloylglycine in step (1) is 1:3-15; the amino acid sequence of the antimicrobial peptide is shown in SEQ ID NO.

1.

4. The method for preparing the oil-controlling, acne-removing, and antibacterial peptide co-amorphous assembly according to claim 1, characterized in that, The mass-to-volume ratio of the antimicrobial peptide to water in step (1) is 1:100-1000 g / ml.

5. The method for preparing the oil-controlling, acne-removing, and antibacterial peptide co-amorphous assembly according to claim 1, characterized in that, The temperature of rotary evaporation in step (1) is 50-70℃; the temperature of drying is 40-60℃, and the drying time is 24-48h.

6. The method for preparing the oil-controlling, acne-removing, and antibacterial peptide co-amorphous assembly according to claim 1, characterized in that, The cyclodextrin compound in step (2) is at least one of β-cyclodextrin, hydroxypropyl-β-cyclodextrin, methyl-β-cyclodextrin, γ-cyclodextrin, sulfobutyl ether-β-cyclodextrin, and hydroxypropyl-α-cyclodextrin; the mass-to-volume ratio of the cyclodextrin compound to water is (1-1.5):(40-60) g / mL; the mass ratio of the cyclodextrin compound to the amorphous antimicrobial peptide is 1:0.5-5.

7. The method for preparing the oil-controlling, acne-removing, and antibacterial peptide co-amorphous assembly according to claim 1, characterized in that, In step (2), the stirring speed is 300-600 rpm; the heating temperature is 40-60℃; and the heating time is 2-6 h.

8. The method for preparing the oil-controlling, acne-removing, and antimicrobial peptide co-amorphous assembly according to claim 1, characterized in that, The inlet temperature of the spray dryer in step (2) is 120-160℃, the outlet temperature of the spray dryer is 60℃-80℃, the feed rate of the spray dryer is 0.2-0.6L / h, and the atomizing gas flow rate is 2-8m³ / h.

9. An amorphous assembly of oil-controlling, acne-removing, and antibacterial peptides prepared by the preparation method according to any one of claims 1-8.

10. The use of the oil-controlling, acne-removing, and antibacterial peptide co-amorphous assembly according to claim 9 in the preparation of topical medicines or cosmetics.

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