Phellodendron amurense alcohol extraction method and application of phellodendron amurense extract as film coating agent

Abstract

The invention provides an alcohol extraction method of cortex phellodendri and application of the cortex phellodendri alcohol extract as a liniment.The invention relates to the technical field of medicine.The alcohol extraction method of cortex phellodendri is provided, the alcohol extract of cortex phellodendri is obtained through the extraction method, the bacteriostatic activity of the alcohol extract of cortex phellodendri on propionibacterium acnes is 1.56 mg / mL, and the bacteriostatic activity of the alcohol extract of cortex phellodendri is remarkably superior to that of other existing traditional Chinese medicine extraction bacteriostatic schemes; meanwhile, the invention provides the golden cypress alcohol extract liniment for inhibiting acnes, propionibacterium acnes are synergistically inhibited by reducing the protein expression levels of Src, Akt and mTOR in skin tissues and destroying the completeness of cell walls and cell membranes of the propionibacterium acnes, and the liniment is not added with a penetration enhancer, does not irritate the skin, is safer to use, and has a good application prospect. The formed medicine film reduces evaporation of moisture on the skin surface, the medicine is promoted to be slowly released through cuticle, an animal model verifies that acnes rapidly fade after 4 days, a non-antibiotic treatment scheme is provided for light and moderate acnes, and a better treatment effect is achieved.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a method for extracting phellodendron alcohol and application of the extract as a coating agent. Background Art

[0002] Acne is a chronic inflammatory skin disease of the sebaceous follicles, affecting nearly 80% of adolescents and young adults. It typically persists into adulthood, typically appearing on the cheeks, forehead, chin, and back. It can cause scarring and hyperpigmentation, and can have a significant negative psychological impact on patients. Studies have shown that from 1900 to 2021, the incidence of acne among adolescents and young adults aged 10-24 years has increased globally, with the highest prevalence among adolescents aged 15-19 years. Acne typically initially manifests as whiteheads or blackheads, caused by the accumulation or embolism of sebum and keratinocytes at the opening of the hair follicle. If bacterial infection occurs after the pores become blocked, they develop into red, raised papules. In severe cases, pustules may form at the top of the papules. The pathogenesis of acne is complex, linked not only to androgen-mediated increased sebaceous gland secretion, altered sebum composition, abnormal keratinization of the sebaceous ducts, the proliferation of Propionibacterium acnes, and an inflammatory response, but also to genetic factors and dietary habits.

[0003] Existing acne treatment products contain permeation enhancer ingredients. Some chemically synthesized permeation enhancers are irritating to the skin. When the concentration of anionic surfactant exceeds the critical micelle concentration, it can change the lipid bilayer structure of the stratum corneum, remove keratin and swell the keratinocyte layer, thereby irritating the skin. Some permeation enhancers may even cause skin allergic reactions.

[0004] Therefore, there is an urgent need to invent a method for extracting phellodendron alcohol and a coating agent prepared from the extract to solve the above-mentioned existing technical problems. Summary of the Invention

[0005] The present invention proposes a method for extracting phellodendron alcohol and the use of the extract as a film-forming agent. The antibacterial concentration of the phellodendron alcohol extract against Propionibacterium acnes is 1.56 mg / mL, which is significantly better than other existing traditional Chinese medicine extraction antibacterial schemes. The film-forming agent of the present invention does not have the phenomenon of drug pigment deposition and does not contain a permeation enhancer. It achieves high permeability through the performance of the drug itself, thereby solving the above-mentioned technical problems.

[0006] To achieve the above objectives, the present invention is implemented through the following technical solutions: A film-forming agent prepared from a Phellodendron amurense alcohol extract comprises the Phellodendron amurense alcohol extract, a film-forming material, a plasticizer, a surfactant and a solvent. The film-forming material is polyvinyl alcohol, the polyvinyl alcohol is PVA22-88, the plasticizer is glycerol, the surfactant is Tween-80, and the solvent is water.

[0007] Preferably, the method for extracting the Phellodendron amurense alcohol extract comprises the following steps: S1: Weigh Phellodendron chinense, soak in ethanol solution, and extract under reflux in a water bath for 2 hours. Collect the filtrate. S2: The filter residue was added with ethanol solution and extracted again for 1.5 h, and the two filtrates were mixed; S3: The mixed filtrate was concentrated using a rotary evaporator, centrifuged at 4000 rpm for 5 min, and then placed in a sterile environment at 4°C overnight; S4: Collect the supernatant after centrifugation and freeze it in a -20°C refrigerator, then transfer it to a freeze dryer and freeze-dry it for 48 hours to form a powder; S5: Take the powder and dilute it with water, pour it into a separatory funnel, add petroleum ether, shake and extract, and let it stand; S6: Collect the upper and lower layer solutions separately, repeat 3 times, and then add ethyl acetate for extraction; S7: Collect the upper and lower layer solutions after extraction and repeat 3 times. Freeze the aqueous layer at -20°C for 8 hours and then put it into a freeze dryer and freeze-dry it for 48 hours to obtain a Phellodendron chinense alcohol extract.

[0008] Preferably, the volume fraction of ethanol in S1 is 70%, the water bath temperature is 70° C., and the volume fraction of ethanol in S2 is 70%.

[0009] Preferably, the berberine content in the Phellodendron chinense ethanol extract is greater than 15%, and the minimum inhibitory concentration of the aqueous phase component of the Phellodendron chinense ethanol extract after purification by petroleum ether-ethyl acetate is 1.56 mg / mL.

[0010] Preferably, the components of the coating agent in mass percentage are: 2-6% of Phellodendron chinense alcohol extract, 8-12% of PVA22-88, 10-20% of glycerol, 1.5-2.5% of Tween-80, and the rest is water.

[0011] Preferably, the film-coating treats acne by inhibiting the Src / Akt / mTOR signaling pathway of Propionibacterium acnes, which is specifically manifested in that the film-coating reduces the expression levels of Src, Akt, and mTOR proteins in skin tissue and destroys the integrity of the cell wall and cell membrane of Propionibacterium acnes, resulting in the leakage of Propionibacterium acnes alkaline phosphatase, nucleic acids, and proteins.

[0012] Preferably, the coating agent is used to conduct an in vitro permeation experiment on rabbit skin, and the in vitro permeation parameter calculation formula of the coating agent is as follows:

[0013] Where: Q is the cumulative permeation amount per unit area, F is the drug release rate, J is the permeation flux, Cd is the drug concentration in the diffusion cell, Vr is the volume of the diffusion cell, A is the cross-sectional area of the diffusion cell, Dose is the drug loading per unit area, and k is the slope.

[0014] Preferably, when the coating agent is selected from the formula of PVA22-8810%, glycerol 10%, Tween-802.5%, and Phellodendron amurense alcohol extract 6%, the cumulative permeation per unit area per 24h in vitro transdermal treatment in the preferred formula of the coating agent is 82.56±10.45μg / cm2 / h.

[0015] Preferably, the components of the Phellodendron chinense alcohol extract include phellodendrine, 3-O-feruloylquinic acid, 5-O-feruloylquinic acid, and berberine.

[0016] Preferably, the method for preparing a film-forming agent from the alcohol extract of Phellodendron amurense comprises the following steps: P1: Add PVA22-88 to distilled water and stir until completely dissolved. After dissolution, the concentration of PVA22-88 is 10%; P2: After cooling to 40-50°C, add glycerol, Tween-80 and Phellodendron amurense alcohol extract in sequence and stir evenly; P3: After standing and degassing, apply it to the mold and dry it for 15-30 minutes to form a film with a thickness of 0.1-0.3mm.

[0017] Beneficial effects: The present invention provides a method for extracting phellodendron alcohol and the use of the extract as a coating agent, which has the following beneficial effects compared with existing technologies: The present invention provides a method for extracting phellodendron alcohol. Using this method, the phellodendron alcohol extract achieved an antibacterial concentration of 1.56 mg / mL against Propionibacterium acnes, significantly outperforming other existing traditional Chinese medicine extraction methods. The film-coated formulation of the present invention exhibits no pigment deposition. Although the liquid solution is orange-yellow in color, it does not deposit on the skin and can be easily washed off with water or hand soap.

[0018] The present invention reduces the expression levels of Src, Akt and mTOR proteins in skin tissue and destroys the integrity of the cell wall and cell membrane of Propionibacterium acnes, thereby causing the leakage of alkaline phosphatase, nucleic acid and protein to synergistically inhibit Propionibacterium acnes.

[0019] The film-coating formulation of the present invention has been orthogonally optimized and has excellent mechanical properties; the transdermal efficiency is greatly improved, and drug delivery is accelerated. The film-coating of the present invention does not add a penetration enhancer, and achieves high permeability through the properties of the drug itself, without causing additional irritation to the skin. When used, it is applied to the affected area to form a drug film to protect the wound surface, and has good wear resistance, is not easy to fall off, and does not require bandaging. The formed drug film reduces the evaporation of moisture on the skin surface and promotes the slow release of drugs through the stratum corneum. Animal models have verified that acne quickly disappears in 4 days, providing a non-antibiotic treatment option for mild to moderate acne and having a better therapeutic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0021] Figure 1 This is a schematic diagram of the coating agent of the present invention in use; Figure 2 Schematic diagram of alkaline phosphatase activity of Propionibacterium acnes after treatment with 0MIC, 1MIC, 2MIC and 4MIC of Phellodendron amurense alcohol extract for 10 hours; Figure 3 The time-kill kinetic curves of Propionibacterium acnes after treatment with 0MIC, 1MIC, 2MIC and 4MIC of the alcohol extract of Phellodendron amurense for 24 hours are shown in the figure; Figure 4 Schematic diagram of the leakage of Propionibacterium acnes after treatment with 0MIC, 1MIC, 2MIC and 4MIC of Phellodendron amurense alcohol extract for 10 hours; Figure 5 Schematic diagram of total Ca2+ leakage of Propionibacterium acnes after treatment with 0MIC, 1MIC, 2MIC and 4MIC of Phellodendron amurense alcohol extract for 10 hours; Figure 6 The scanning electron microscopic observations of Propionibacterium acnes after treatment with 0MIC, 1MIC, 2MIC and 4MIC of the alcohol extract of Phellodendron amurense for 10 hours are shown in the figure; Figure 7 Schematic diagram of epidermal changes and drug recovery after injection of Propionibacterium acnes; Figure 8 Schematic diagram of the pathological tissue sections of the acne model and the recovery of mice after drug administration in the present invention; Figure 9 This is a grayscale diagram of the Src / Akt / mTOR pathway of the present invention; Figure 10Schematic diagram of the specificity of the in vitro transdermal permeation of the acne model of the present invention; Figure 11 Schematic diagram of the in vitro transdermal results of prescriptions 1 to 3 of the present invention; Figure 12 This is a berberine determination curve diagram of the present invention; Figure 13 This is a diagram demonstrating the efficacy test results of the Phellodendron chinense alcohol extract film-coating agent of the present invention. DETAILED DESCRIPTION

[0022] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention more clearly understood, the present invention is further described in detail in conjunction with the embodiments and drawings. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. The technical solutions of the present invention are described in detail below in conjunction with the embodiments and drawings, but the scope of protection is not limited thereto.

[0023] See also Figure 1-13 , the present invention provides a technical solution: A method for extracting phellodendron alcohol is proposed, comprising the following steps: S1: Weigh Phellodendron chinense, soak in ethanol solution, and extract under reflux in a water bath for 2 hours. Collect the filtrate. S2: The filter residue was added with ethanol solution and extracted again for 1.5 h, and the two filtrates were mixed; S3: The mixed filtrate was concentrated using a rotary evaporator, centrifuged at 4000 rpm for 5 min, and then placed in a sterile environment at 4°C overnight; S4: Collect the supernatant after centrifugation and freeze it in a -20°C refrigerator, then transfer it to a freeze dryer and freeze-dry it for 48 hours to form a powder; S5: Take the powder and dilute it with water, pour it into a separatory funnel, add petroleum ether, shake and extract, and let it stand for 15 minutes; S6: Collect the upper and lower layer solutions separately, repeat 3 times, and then add ethyl acetate for extraction; S7: Collect the upper and lower layer solutions after extraction and repeat 3 times. Freeze the aqueous layer at -20°C for 8 hours and then put it into a freeze dryer and freeze-dry it for 48 hours to obtain a Phellodendron chinense alcohol extract.

[0024] The specific experiments are: Ethanol extraction of Phellodendron chinense: Weigh 50g of Phellodendron chinense and soak it in 350mL of 70% ethanol. Extract it in a 70℃ water bath under reflux for 2 hours. Collect the filtrate and add 400mL of 70% ethanol to the residue for another 1.5 hours. Combine the two filtrates and concentrate the extracted filtrates using a rotary evaporator. Centrifuge them at 4000rpm for 5 minutes and then let them stand at 4℃ overnight. After centrifugation the next day, collect the supernatant and freeze it in a -20℃ refrigerator. Transfer it to a freeze dryer and lyophilize it for 48 hours to powder.

[0025] Water decoction extraction of Phellodendron chinense: weigh 50g of Phellodendron chinense, add 500mL of distilled water respectively, soak for 1.5h, extract twice, each time for 2h, combine the two filtrates, use rotary evaporator to concentrate to 500mL, pour in a certain volume of 95% ethanol to make the alcohol concentration of 70%, place at 4℃ overnight, centrifuge the liquid at 4000rpm for 5min, collect the supernatant and freeze it, put it in freeze dryer for 48h, and make it into powder for later use.

[0026] The experiment measured that the extraction rate of Phellodendron chinense alcohol extract was 12.8%, and the water extraction rate was 12%. In this application, Kochia scoparia, Humulus, Cnidium monnieri, Taraxacum officinale, and Sophora flavescens were further extracted with alcohol and water, and equal amounts of extracts were taken for Propionibacterium acnes antibacterial test. The test results are shown in Table 1. The antibacterial concentration of the alcohol extract of Phellodendron chinense was 3.12 mg / mL, and the antibacterial effect was better than that of other traditional Chinese medicines.

[0027] Table 1: Minimum inhibitory concentration of various Chinese herbal medicine extracts

[0028] The alcohol extract of Phellodendron amurense was further purified by weighing 1 g of Phellodendron amurense extract powder, adding 20 mL of distilled water as a dispersant, pouring it into a separating funnel, and then adding 20 mL of petroleum ether for oscillation extraction. After waiting for 15 minutes, the upper and lower layers of the solution were collected separately, shaken and then layered statically. This was repeated 3 times and mixed and released to obtain a darker petroleum ether extraction upper layer and a lighter aqueous solution lower layer. 20 mL of ethyl acetate was added for extraction. The upper and lower layers of the solution were collected separately, shaken and then layered statically. After this was repeated 3 times, the upper layer was the ethyl acetate layer and the lower layer was the aqueous layer. The aqueous layer was frozen at -20°C for 8 hours and then placed in a freeze dryer for freezing. The extracts were dried for 48 hours, dried and concentrated under reduced pressure on a rotary evaporator, the petroleum ether and ethyl acetate were recovered, and the remaining ethyl acetate layer and petroleum ether layer were placed in a vacuum drying oven at 40°C for drying. The remaining trace amount of petroleum ether and ethyl acetate as well as any water present in the organic solvent were further removed. The purified alcohol extract of Phellodendron amurense was subjected to an antibacterial test on Propionibacterium acnes. After drying, petroleum ether was in a viscous liquid state and could not be dissolved in water. Therefore, the extraction rate and minimum inhibitory concentration experimental results could not be obtained. The results are shown in Table 2 below. The antibacterial concentration of the purified yellow powder of the water layer was 1.56 mg / ml.

[0029] Table 2: Antibacterial test results of purified Phellodendron amurense alcohol extract

[0030] In some embodiments, a time-disinfecting curve assay was performed, and Propionibacterium acnes in the logarithmic growth phase was washed three times with phosphate buffer, the turbidity was adjusted to 0.5 using a turbidimetric tube, and 0MIC, 1MIC, 2MIC, and 4MIC alcohol extracts of Phellodendron amurense were added to an equal volume of bacterial solution. 100 μL of sample was taken every 2 hours, and gradient dilution was performed in LB broth plate solid culture medium, and cultured in an anaerobic environment at 37°C for 48 hours.

[0031] The time-kill curve of the alcohol extract of Phellodendron amurense was determined by culturing it with Propionibacterium acnes for 24 hours. The results showed that the colony count of the 0MIC group did not change significantly after 24 hours of culture. Compared with 0MIC, the colony count was significantly reduced after 12 hours of culture when 1MIC and 2MIC Phellodendron amurense alcohol extract solutions were added. The colony count of the Phellodendron amurense alcohol extract with 4MIC of Phellodendron amurense alcohol extract was significantly reduced at 2 hours. Both low and high concentrations of Phellodendron amurense alcohol extract can significantly inhibit the growth of P. acnes, and the effect is dose-dependent.

[0032] In some embodiments, alkaline phosphatase activity was measured and a time-disinfect curve experiment was performed. P. acnes in the logarithmic growth phase was washed three times with phosphate buffer and the turbidity was adjusted to 0.5 using a turbidimetric tube. The alcohol extract of P. acnes in the 0 MIC and 1 MIC groups was incubated with P. acnes under anaerobic conditions at 37°C for 10 hours. Samples were taken at 0, 2, 4, 6, 8, and 10 hours, centrifuged at 8000 g for 10 minutes, and the supernatant was collected. The alkaline phosphatase activity was measured using an alkaline phosphatase detection kit (Nanjing Jiancheng Biological Research Institute). The alkaline phosphatase activity of P. acnes in the 0 MIC group remained almost unchanged after 10 hours of incubation. Compared with the 0 MIC group, the alkaline phosphatase activity of P. acnes in the presence of the alcohol extract of P. acnes increased significantly, and the activity increased significantly with increasing concentrations of the alcohol extract of P. acnes.

[0033] The effect of Phellodendron amurense alcohol extract on the permeability of Propionibacterium acnes was determined. P. acnes were collected during the logarithmic growth phase, washed three times with phosphate buffered saline, and then suspended in phosphate buffered saline. The bacterial suspension was adjusted to a McFarland turbidity of 0.5. Phellodendron amurense alcohol extracts with 0 and 1 MIC groups were incubated with P. acnes at 37°C under anaerobic conditions for 10 hours. Samples were taken at 0, 2, 4, 6, 8, and 10 hours, and the leakage of nucleic acids and proteins was measured using a UV spectrophotometer at 260 nm and 280 nm. Ca 2+ The assay steps are the same as for nucleic acids and proteins, using Ga 2+ Kit detection of Ga 2+ Ion content.

[0034] Nucleic acids and proteins have maximum absorption peaks at 260 nm and 280 nm, respectively. The absorption intensity is proportional to their concentration. The concentrations of nucleic acids and proteins in the extracellular fluid of P. acnes were measured using ultraviolet absorption. After incubating P. acnes with various concentrations of Phellodendron amurense ethanol extract for 10 hours, the concentrations of nucleic acids and proteins increased significantly at the second hour, and continued to increase significantly with increasing Phellodendron amurense ethanol extract concentration.

[0035] Ga 2+ Also exists in the cell membrane, so Ga 2+ The leakage of Ga in the 0MIC group also indicates that the bacterial cell membrane is damaged. 2+ The concentration change was not obvious. After 10 hours of culture with Propionibacterium acnes and Phellodendron amurense ethanol extract, the Ca 2+ The concentration increased significantly at the 2nd hour and then stabilized. Therefore, the alcohol extract of Phellodendron amurense can destroy the cell wall and cell membrane of Propionibacterium acnes to kill the bacteria.

[0036] In some examples, 8-week-old male ICR mice (weighing 30-32 g) were purchased from Liaoning Changsheng Laboratory Animal Technology Co., Ltd. After one week of adaptive feeding, the mice were randomly divided into six groups: blank control (CON), model group (MOD), adapalene group (Ada), low-dose group (1MIC), medium-dose group (2MIC), and high-dose group (4MIC). The recovery of mice in each treatment group was recorded and photographed. Compared with the CON group, mice in the model group developed cysts and papules on the abdominal surface, accompanied by ulceration and scabs, after injection with Propionibacterium acnes. Abdominal symptoms in the Ada, 1MIC, 2MIC, and 4MIC groups were similar to those in the MOD group before treatment. After one week of Ada treatment, symptoms remained unchanged, similar to those in the MOD group. After five days of topical treatment with a Phellodendron amurense alcohol extract solution, abdominal lesions in all groups improved to varying degrees. Abdominal inflammation in the 2MIC and 4MIC groups was significantly alleviated, with abscesses largely resolved. Abdominal inflammation and abscesses in the 1MIC group were slightly reduced, with some resolution compared to pre-treatment levels.

[0037] HE staining of the abdominal skin of mice showed an abundance of hair follicles, sebaceous glands, and other skin appendages in the dermis of the CON group, with occasional lymphocytes. No obvious hyperkeratosis or other abnormalities were observed. Compared with the CON group, the MOD group showed a blurred dermal-subcutaneous boundary, extensive connective tissue hyperplasia, neovascularization, and infiltration of numerous lymphocytes and granulocytes, with swollen cells and loose cytoplasm. Compared with the MOD group, connective tissue, lymphocytes, and granulocytes decreased in all treatment groups, with the 4MIC group showing more significant recovery from abdominal inflammation.

[0038] Western blot analysis of related protein expression in skin tissue revealed that Phellodendron amurense ethanol extract can inhibit cortical secretion by downregulating Src, Akt, and mTOR proteins, thereby achieving the goal of treating acne. Phellodendron amurense ethanol extract can treat acne by inhibiting the Src / Akt / mTor pathway. The CON group showed lower expression levels of Src, Akt, and mTor proteins than the other groups. Compared with the CON group, the MOD group showed significantly increased expression levels of Src, Akt, and mTor proteins. Compared with the MOD group, the expression levels of Src, Akt, and mTor in skin tissue of each treatment group decreased to varying degrees.

[0039] Phellodendron amurense alcohol extract can destroy the cell wall and membrane of Propionibacterium acnes, thereby killing the bacteria. Time-disinfect curve results showed that both low and high concentrations of Phellodendron amurense alcohol extract significantly inhibited the growth of P. acnes in a dose-dependent manner. Western blot results showed that Phellodendron amurense alcohol extract can inhibit cortical secretion by downregulating Src, Akt, and mTOR proteins, thereby achieving the goal of treating acne.

[0040] In some embodiments, the berberine content in the Phellodendron chinense ethanol extract was measured, and the average berberine content was found to be 15%.

[0041] In some embodiments, by combining mass spectrometry total ion current and ultraviolet absorption chromatogram analysis, a total of 7 compounds were found in the transdermal skin samples as shown in Table 3 below: Table 3: Components of Phellodendron amurense alcohol extract that penetrate the skin

[0042] In some embodiments, a film-coating agent was prepared using an alcohol extract of Phellodendron chinense. Appropriate amounts of PVA 22-88 and PVA 35-80 were added to distilled water; appropriate amounts of PVA 574 and PVA L-508w were added to 50% ethanol, and all were dissolved to a concentration of 10%. A single-factor experiment was used to investigate the effects of adding different concentrations of glycerol, Tween-80, and different drug doses on tensile strength and elongation, thereby screening a suitable prescription range. The single-factor analysis is shown in Table 4.

[0043] Table 4: Univariate analysis table

[0044] On the basis of single-factor experiments, SPSS was used to conduct orthogonal experimental design with glycerol, Tween-80 and drug loading as investigation factors and tensile strength, elongation and film-forming time as indicators. The orthogonal experimental design is shown in Table 5.

[0045] Table 5: Orthogonal experimental design table

[0046] In some embodiments, PVA with 5% glycerol is used as the initial formula, and glycerol is added in gradients of 5%, 10%, 15%, 20% and 25% to measure the elongation and tensile strength of four types of PVA. Because PVA 574 and PVA L-508W have poor film-forming properties when 25% glycerol is added, the films are too thin and soft, so their elongation and tensile strength cannot be measured. Compared with the initial formula, the elongation of PVA22-88 and PVA 35-80 is the largest when the glycerol content is 15%, and the tensile strength is the largest when the glycerol content is 5%; the elongation of PVA 574 and PVA L-508W is the largest when the glycerol content is 10%, and the tensile strength is the largest when the glycerol content is 5%. The tensile strength of the four PVAs decreases with increasing glycerol content. Taking both indicators into consideration, the mechanical strength of PVA 22-88 and PVA 35-80 was the best when the glycerol addition concentration was 15%, while the mechanical strength of PVA 574 and PVA L-508W was the best when the glycerol addition concentration was 10%.

[0047] In some embodiments, the glycerol concentration of PVA 22-88 and PVA 35-80 is set to a fixed value of 15%, the glycerol concentration of PVA 574 and PVA L-508W is set to a fixed value of 10%, and Tween-80 is added in gradients of 0.5%, 1%, 1.5%, 2% and 2.5% to measure their elongation and mechanical properties. After adding Tween-80, except for PVA 35-80, the elongation of the other three PVAs after adding 0.5% Tween-80 is significantly reduced. Compared with the addition of 0.5% Tween-80, the elongation of PVA 22-88 is significantly increased after adding 2.5% Tween-80. The addition of Tween-80 has no significant effect on the tensile strength of PVA. Taking all factors into consideration, the Tween-80 addition concentration of PVA 22-88, PVA35-80 and PVAL-508W is 2%, and that of PVA 574 is 1%. At this time, the mechanical properties of the four PVAs are most similar.

[0048] Different glycerol and Tween-80 concentrations and drug loadings had varying effects on the mechanical properties of PVA. Glycerol concentration and drug loading had a more pronounced effect on elongation, while Tween-80 had a lesser effect. All four PVA types were capable of forming film-forming agents under the experimental conditions. PVA 22-88 exhibited greater tensile strength and superior elongation than the other PVAs, and since various experimental factors significantly influenced its properties, PVA 22-88 was selected as the subsequent drug-loading matrix.

[0049] Orthogonal tests were conducted with different glycerol, Tween-80 concentrations, and drug loadings. The test results are shown in Table 6 below. Combined with the orthogonal test results and range analysis, the orthogonal test was used to screen the formulations of the film-coating agent of the alcohol extract of Phellodendron amurense. Three formulations were determined based on elongation, tensile strength, and film-forming time as indicators: Prescription 1: PVA 22-88 accounts for 10% by weight, glycerin accounts for 10% by weight, Tween accounts for 2.5% by weight, and Phellodendron chinense alcohol extract accounts for 6% by weight; Prescription 2: PVA 22-88 accounts for 10% by weight, glycerin accounts for 20% by weight, Tween accounts for 1.5% by weight, and Phellodendron chinense alcohol extract accounts for 6% by weight; Prescription 3: PVA 22-88 accounts for 10% by mass, glycerin accounts for 10% by mass, Tween accounts for 1.5% by mass, and Phellodendron chinense alcohol extract accounts for 2% by mass.

[0050] Table 6: Results of orthogonal experiments on glycerol, Tween-80 concentration, PVA and drug loading

[0051] In some embodiments, the steps for preparing the Phellodendron amurense alcohol extract film-coating agent include: P1: Add PVA22-88 to distilled water and stir until completely dissolved. After dissolution, the concentration of PVA22-88 is 10%; P2: After cooling to 40-50°C, add glycerol, Tween-80 and Phellodendron amurense alcohol extract in sequence and stir evenly; P3: After standing and degassing, apply it to the mold and dry it for 15-30 minutes to form a film with a thickness of 0.1-0.3mm.

[0052] In some embodiments, rabbit skin was mounted in a vertical diffusion cell (diffusion area = 1.77 cm 2 ) was evenly applied to the stratum corneum of the skin using a 0.2 ml film of Phellodendron amurense alcohol extract. The diffusion cell contained 4 mL of phosphate-buffered saline receptor solution, and the stirrer was maintained at 600 rpm. The temperature was maintained at 32 ± 0.5°C. Samples were extracted at 2, 4, 6, 8, 12, and 24 hours, and fresh receptor solution was immediately added to maintain equilibrium in the cell. Samples were analyzed by HPLC. All in vitro experiments were repeated three times, and the cumulative drug permeation per unit area, Q, was plotted as a function of time t.

[0053] HPLC chromatography was performed using a Welch Ultimate® LP-C18 column (4.6×250 mm, 5 μm). The mobile phases were acetonitrile (A) and 0.1% phosphoric acid solution (B). The detection wavelength was 345 nm, the column temperature was 30°C, the flow rate was 1 mL / min, and the gradient elution program was as shown in Table 7: Table 7: Transdermal Chromatography Conditions and Elution Schedule

[0054] The calculation formula of in vitro transdermal parameters is as follows:

[0055] Where: Q is the cumulative permeation amount per unit area, F is the drug release rate, J is the permeation flux, Cd is the drug concentration in the diffusion cell, Vr is the volume of the diffusion cell, A is the cross-sectional area of the diffusion cell, Dose is the drug loading per unit area, and k is the slope.

[0056] Three prescriptions were screened based on tensile strength, elongation, and film-forming time. The final screening was performed based on the in vitro transdermal results. The transdermal parameter calculation results are shown in Table 8. After comparison, prescription 1 was selected as the best prescription for subsequent efficacy tests.

[0057] Table 8: Calculation results of transdermal parameters of Phellodendron amurense ethanol extract

[0058] Therefore, the main components of the film-coating agent of the present invention are 2-6% of the alcohol extract of Phellodendron chinense, 8-12% of PVA 22-88, 10-20% of glycerol, 1.5-2.5% of Tween-80, and the rest is water. When the mass proportion of PVA 22-88 is 10%, the mass proportion of glycerol is 10%, the mass proportion of Tween is 2.5%, and the mass proportion of Phellodendron chinense alcohol extract is 6%, the best transdermal experimental effect is achieved.

[0059] In some embodiments, efficacy tests showed that after 5 consecutive days of administration, without the use of any penetration enhancers, the mice in the group treated with the film prepared from the alcohol extract of Phellodendron amurense had basically recovered on the 4th day, while the model group still had cysts, indicating that this film has the effect of treating acne.

[0060] The above content is a further detailed description of the present invention in combination with specific preferred embodiments. For ordinary technicians in the technical field to which the present invention belongs, they can make several simple deductions or substitutions without departing from the present invention, which should be regarded as belonging to the scope of patent protection determined by the submitted claims of the present invention.

Claims

1. A film-coating agent prepared from an alcohol extract of Phellodendron amurense, characterized in that: The invention comprises phellodendron amurense alcohol extract, film-forming material, plasticizer, surfactant and solvent. The film-forming material is polyvinyl alcohol, the polyvinyl alcohol is PVA22-88, the plasticizer is glycerol, the surfactant is Tween-80, and the solvent is water.

2. The film-coating agent prepared from the alcohol extract of Phellodendron amurense according to claim 1, characterized in that: The method for extracting the Phellodendron amurense alcohol extract comprises the following steps: S1: Weigh Phellodendron chinense, soak in ethanol solution, and extract under reflux in a water bath for 2 hours. Collect the filtrate. S2: The filter residue was added with ethanol solution and extracted again for 1.5 h, and the two filtrates were mixed; S3: The mixed filtrate was concentrated using a rotary evaporator, centrifuged at 4000 rpm for 5 min, and then placed in a sterile environment at 4°C overnight; S4: Collect the supernatant after centrifugation and freeze it in a -20°C refrigerator, then transfer it to a freeze dryer and freeze-dry it for 48 hours to form a powder; S5: Take the powder and dilute it with water, pour it into a separatory funnel, add petroleum ether, shake and extract, and let it stand; S6: Collect the upper and lower layer solutions separately, repeat 3 times, and then add ethyl acetate for extraction; S7: Collect the upper and lower layer solutions after extraction and repeat 3 times. Freeze the aqueous layer at -20°C for 8 hours and then put it into a freeze dryer and freeze-dry it for 48 hours to obtain a Phellodendron chinense alcohol extract.

3. The film-coating agent prepared from the alcohol extract of Phellodendron amurense according to claim 2, characterized in that: The volume fraction of ethanol in the S1 is 70%, the water bath temperature is 70° C., and the volume fraction of ethanol in the S2 is 70%.

4. The film-coating agent prepared from the alcohol extract of Phellodendron amurense according to claim 2, characterized in that: The berberine content in the Phellodendron chinense alcohol extract is greater than 15%, and the minimum inhibitory concentration of the aqueous phase component of the Phellodendron chinense alcohol extract after purification by petroleum ether-ethyl acetate is 1.56 mg / mL.

5. The film-coating agent prepared from the alcohol extract of Phellodendron amurense according to claim 1, characterized in that: The mass percentage components of the coating agent are: 2-6% of Phellodendron amurense alcohol extract, 8-12% of PVA22-88, 10-20% of glycerol, 1.5-2.5% of Tween-80, and the rest of water.

6. The film-coating agent prepared from the alcohol extract of Phellodendron amurense according to claim 1, characterized in that: The coating treats acne by inhibiting the Src / Akt / mTOR signaling pathway of Propionibacterium acnes. Specifically, the coating reduces the expression levels of Src, Akt, and mTOR proteins in skin tissue and destroys the integrity of the cell wall and cell membrane of Propionibacterium acnes, resulting in the leakage of Propionibacterium acnes alkaline phosphatase, nucleic acids, and proteins.

7. The film-coating agent prepared from the alcohol extract of Phellodendron amurense according to claim 1, characterized in that: The coating agent was used to conduct an in vitro permeation experiment on rabbit skin. The in vitro permeation parameter calculation formula of the coating agent is as follows: ； Where: Q is the cumulative permeation amount per unit area, F is the drug release rate, J is the permeation flux, Cd is the drug concentration in the diffusion cell, Vr is the volume of the diffusion cell, A is the cross-sectional area of the diffusion cell, Dose is the drug loading per unit area, and k is the slope.

8. The film-coating agent prepared from the alcohol extract of Phellodendron amurense according to claim 1, characterized in that: When the coating agent is selected with a ratio of 10% by weight of PVA22-88, 10% by weight of glycerol, 2.5% by weight of Tween-80, and 6% by weight of Phellodendron amurense alcohol extract, the cumulative permeation per unit area of the coating agent in vitro for 24 hours is 82.56±10.45 μg / cm 2 / h.

9. The film-coating agent prepared from the alcohol extract of Phellodendron amurense according to any one of claims 1 to 8, characterized in that: The components of the phellodendron chinense alcohol extract include phellodendronine, 3-O-feruloylquinic acid, 5-O-feruloylquinic acid and berberine.

10. A method for preparing a film-forming agent from a Phellodendron amurense alcohol extract, characterized in that: The following steps are involved: P1: Add PVA22-88 to distilled water and stir until completely dissolved. After dissolution, the concentration of PVA22-88 is 10%; P2: After cooling to 40-50°C, add glycerol, Tween-80 and Phellodendron amurense alcohol extract in sequence and stir evenly; P3: After standing and degassing, apply it to the mold and dry it for 15-30 minutes to form a film with a thickness of 0.1-0.3mm.

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