Extraction method of phellodendri chinensis and application of the extract as a coating agent

Abstract

The application discloses a phellodendron amurense Rupreth alcohol extraction method and application of the phellodendron amurense Rupreth alcohol extract as a film coating agent, and relates to the technical field of medicines.The application provides a phellodendron amurense Rupreth alcohol extraction method, and the phellodendron amurense Rupreth alcohol extract is obtained through the extraction method.The bacteriostatic activity of the obtained phellodendron amurense Rupreth alcohol extract on propionibacterium acnes is 1.56 mg / mL, which is significantly better than that of other existing traditional Chinese medicine extraction bacteriostatic schemes.Meanwhile, the application provides a phellodendron amurense Rupreth alcohol extract film coating agent for inhibiting acne, which can synergistically inhibit propionibacterium acnes by reducing the expression levels of Src, Akt and mTOR proteins in skin tissues and destroying the cell wall and cell membrane integrity of propionibacterium acnes.The film coating agent does not add a penetration enhancer, and thus does not irritate the skin, is safer to use, reduces the evaporation of skin surface moisture, promotes the slow release of the drug through the stratum corneum, and verifies the rapid regression of acne in an animal model in four days, thereby providing a non-antibiotic treatment scheme for mild and moderate acne and better playing a therapeutic role.

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, involving not only androgen-mediated increased sebaceous gland secretion, altered sebum composition, abnormal keratinization of the sebaceous ducts, the proliferation of Propionibacterium acnes, and inflammatory responses, but also genetic factors and dietary habits as significant contributors.

[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:

[0007] 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.

[0008] Preferably, the extraction method of the phellodendri chinensis alcohol extract comprises the following steps:

[0009] S1: weigh the phellodendri chinensis, add ethanol solution for soaking, water bath reflux extraction, 2h for the first time, collect the filtrate;

[0010] S2: add ethanol solution to the residue for the second extraction for 1.5h, and mix the filtrates of the two times;

[0011] S3: concentrate the mixed filtrate using a rotary evaporator, centrifuge at 4000rpm for 5min, and then place it in a 4℃ sterile environment for overnight;

[0012] S4: collect the supernatant after centrifugation, freeze it in a-20℃ refrigerator, and then freeze dry it in a freeze dryer for 48h to form a powder;

[0013] S5: take the powder, dilute it with water, pour it into a separatory funnel, add petroleum ether, shake and extract, and then stand still;

[0014] S6: collect the upper and lower layer solutions respectively, repeat 3 times, and then extract with ethyl acetate;

[0015] S7: collect the upper and lower layer solutions after extraction and repeat 3 times, freeze the water layer in-20℃ for 8h, and then freeze dry it in a freeze dryer for 48h to form a powder, thereby obtaining the phellodendri chinensis alcohol extract.

[0016] Preferably, the volume fraction of ethanol in S1 is 70%, and the water bath temperature is 70℃.

[0017] Preferably, the content of berberine in the phellodendri chinensis alcohol extract is greater than 15%, and the minimum inhibitory concentration of the water phase component after purification of the phellodendri chinensis alcohol extract with petroleum ether-ethyl acetate is 1.56mg / mL.

[0018] Preferably, the mass percentage components of the film coating agent are as follows: 2-6% of the phellodendri chinensis alcohol extract, 8-12% of PVA22-88, 10-20% of glycerol, 1.5-2.5% of Tween-80, and the rest is water.

[0019] Preferably, the film coating agent treats acne by inhibiting the Src / Akt / mTOR signal pathway of propionibacterium acnes, specifically by reducing the expression levels of Src, Akt, and mTOR proteins in skin tissue and damaging the cell wall and membrane integrity of propionibacterium acnes, thereby causing leakage of alkaline phosphatase, nucleic acid, and protein of propionibacterium acnes.

[0020] Preferably, the film coating agent is used for in vitro transdermal experiment on rabbit skin, and the in vitro transdermal parameter calculation formula of the film coating agent is as follows:

[0021]

[0022] In the formula: Q is the cumulative permeation 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.

[0023] Preferably, the film coating agent is selected from the PVA22-88 10%, glycerol 10%, Tween-80 2.5%, and Cortex Phellodendri extract 6% scheme, and the cumulative permeation per unit area of the film coating agent in the preferred scheme is 82.56 ± 10.45 μg / cm2 / h for 24 hours.

[0024] Preferably, the Cortex Phellodendri extract includes Phellodendrine, 3-O-feruloylquinic acid, 5-O-feruloylquinic acid, and Berberine.

[0025] Preferably, the preparation method of the Cortex Phellodendri extract film coating agent comprises the following steps:

[0026] P1: Add PVA22-88 to distilled water and stir until completely dissolved, and the concentration of PVA22-88 after dissolution is 10%;

[0027] P2: After cooling to 40-50℃, add glycerol, Tween-80, and Cortex Phellodendri extract in sequence and stir uniformly;

[0028] P3: After standing and defoaming, coat in a mold, dry for 15-30 min, and form a film with a thickness of 0.1-0.3 mm.

[0029] Beneficial effects: The Cortex Phellodendri extract method and the application of the extract as a film coating agent provided by the present application have the following beneficial effects compared with existing technologies:

[0030] The Cortex Phellodendri extract method provided by the present application has a bacteriostatic concentration of 1.56 mg / mL for Propionibacterium acnes, which is significantly better than other existing traditional Chinese medicine extraction bacteriostatic schemes, and the film coating agent does not cause drug pigment deposition. Although the color of the drug solution is orange yellow, it does not deposit on the skin and can be easily washed off with water and hand sanitizer.

[0031] In the present application, the expression level of Src, Akt, and mTOR proteins in the skin tissue is reduced, the integrity of the cell wall and cell membrane of Propionibacterium acnes is destroyed, and alkaline phosphatase, nucleic acid, and protein leakage are caused to synergistically inhibit Propionibacterium acnes.

[0032] The prescription of the coating film agent is optimized by an orthogonal experiment, and the mechanical property is excellent; the transdermal efficiency is greatly improved, and the drug delivery is accelerated, the coating film agent does not add a penetration enhancer, high permeability is realized through the performance of the drug itself, no additional irritation is caused to the skin, when used, the coating film agent is coated on the affected area to form a drug film to protect the wound, and the wear resistance is good, and the drug film is not easy to fall off, and the wound does not need to be wrapped, the drug film reduces the evaporation of water on the skin surface, promotes the slow release of the drug through the stratum corneum, and the animal model verifies that the acne is quickly subsided in 4 days, a non-antibiotic treatment scheme is provided for mild and moderate acne, and a better treatment effect is achieved. BRIEF DESCRIPTION OF DRAWINGS

[0033] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or the prior art description. Obviously, the drawings in the following description are only embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of the provided drawings.

[0034] Figure 1 It is a schematic diagram of the use state of the coating film agent of the present application;

[0035] Figure 2 It is a schematic diagram of the alkaline phosphatase activity of Propionibacterium acnes treated with 0MIC, 1MIC, 2MIC and 4MIC Phellodendri Chinensis Cortex ethanol extract for 10h;

[0036] Figure 3 It is a time killing kinetics curve diagram of Propionibacterium acnes treated with 0MIC, 1MIC, 2MIC and 4MIC Phellodendri Chinensis Cortex ethanol extract for 24h;

[0037] Figure 4 It is a content leakage schematic diagram of Propionibacterium acnes treated with 0MIC, 1MIC, 2MIC and 4MIC Phellodendri Chinensis Cortex ethanol extract for 10h;

[0038] Figure 5 It is a total Ca2+ leakage schematic diagram of Propionibacterium acnes treated with 0MIC, 1MIC, 2MIC and 4MIC Phellodendri Chinensis Cortex ethanol extract for 10h;

[0039] Figure 6 It is a scanning electron microscope observation diagram of Propionibacterium acnes treated with 0MIC, 1MIC, 2MIC and 4MIC Phellodendri Chinensis Cortex ethanol extract for 10h;

[0040] Figure 7 It is a schematic diagram of the epidermal changes and drug recovery of Propionibacterium acnes injection skin;

[0041] Figure 8 It is a schematic diagram of pathological tissue sections of acne models and recovery of mice after drug administration in the present application;

[0042] Figure 9 The Src / Akt / mTOR pathway gray value diagram of the present application;

[0043] Figure 10 The specificity diagram of the in vitro transdermal of the acne model of the present application;

[0044] Figure 11 The in vitro transdermal result diagram of the present application in the prescription one to three;

[0045] Figure 12 The berberine determination curve diagram of the present application;

[0046] Figure 13 The efficacy test result demonstration diagram of the cortex phellodendri alcohol extract film coating agent of the present application. DETAILED DESCRIPTION

[0047] In order to make the technical problems to be solved by the present application, the technical solutions and beneficial effects more clear and obvious, the present application is further described in detail in combination with embodiments and drawings. It should be understood that the specific embodiments described herein are only used to explain the present application and do not limit the present application. The technical solutions of the present application are described in detail below in combination with embodiments and drawings, but the protection scope is not limited thereto.

[0048] Please refer to Figures 1-13 The present application provides a technical solution:

[0049] The cortex phellodendri alcohol extraction method is proposed, which comprises the following steps:

[0050] S1: weigh the cortex phellodendri, add ethanol solution for soaking, water bath reflux extraction, 2h for the first time, collect the filtrate;

[0051] S2: add ethanol solution to the residue for the second time extraction for 1.5h, and mix the two filtrates;

[0052] S3: concentrate the mixed filtrate using a rotary evaporator, centrifuge in a centrifuge at a speed of 4000rpm for 5min, and then place it in a 4℃ sterile environment for overnight standing;

[0053] S4: collect the supernatant after centrifugation and freeze it in a-20℃ refrigerator, and then transfer it to a freeze dryer for freeze drying for 48h to form a powder;

[0054] S5: take the powder and dilute it with water, pour it into a separatory funnel, and then add petroleum ether and shake to extract, and stand for 15min;

[0055] S6: collect the upper and lower solutions respectively, repeat 3 times, and then add ethyl acetate for extraction;

[0056] S7: Collect the upper and lower layer solutions after extraction and repeat 3 times, freeze the water layer at -20℃ for 8h and then put it into a freeze dryer to freeze dry for 48h to form a powder, to obtain the extract of Phellodendri chinensis.

[0057] The specific experiment is as follows:

[0058] Phellodendri chinensis ethanol extraction: 50g of Phellodendri chinensis was weighed, soaked in 350mL of 70% ethanol, and extracted by refluxing in a 70℃ water bath, 2h for the first time, the filtrate was collected, and the filter residue was added with 400mL of 70% ethanol for the second time of extraction for 1.5h, the two times of filtrate were combined, the filtrate after extraction was concentrated by using a rotary evaporator, centrifuged in a centrifuge at 4000rpm for 5min, and then placed in 4℃ for overnight. The supernatant was collected after centrifugation the next day, frozen in a -20℃ refrigerator, and then transferred to a freeze dryer for freeze drying for 48h to form a powder.

[0059] Phellodendri chinensis water decoction extraction: 50g of Phellodendri chinensis was weighed, added with 500mL of distilled water respectively, soaked for 1.5h, extracted for 2 times, 2h for each time, the two times of filtrate were combined, concentrated to 500mL by using a rotary evaporator, poured into a certain volume of 95% ethanol to make the alcohol concentration 70%, placed in 4℃ overnight, the supernatant was collected after centrifugation at 4000rpm for 5min and frozen, and then put into a freeze dryer for 48h to form a powder for standby.

[0060] The experimental measurement showed that the extraction rate of Phellodendri chinensis ethanol extract was 12.8%, and the water extraction rate was 12%. In this application, the same amount of Semen Vaccariae, Humulus scandens, Cnidium monnieri, Taraxacum mongolicum and Sophora flavescens were further extracted by ethanol and water, and the same amount of extract was used for acne propionibacterium bacteriostatic test, and the test results are shown in Table 1. The bacteriostatic concentration of Phellodendri chinensis ethanol extract is 3.12mg / mL, and the bacteriostatic effect is better than that of other Chinese medicines.

[0061] Table 1: Minimum bacteriostatic concentration of each Chinese medicine extract

[0062]

[0063] The extract of phellodendri chinensis was further purified. 1 g of the extract of phellodendri chinensis powder was added to 20 mL of distilled water as a dispersant, poured into a separatory funnel, and then 20 mL of petroleum ether was added for oscillation extraction. After 15 min, the upper and lower layers of the solution were collected, shaken, and then allowed to separate. This process was repeated three times, and the resulting mixture was allowed to degas. The upper layer of the petroleum ether extraction had a darker color, and the lower layer of the aqueous solution had a lighter color. 20 mL of ethyl acetate was added for extraction, and the upper and lower layers of the solution were collected, shaken, and then allowed to separate. This process was repeated three times. The upper layer was the ethyl acetate layer, and the lower layer was the water layer. The water layer was frozen at -20°C for 8 h and then placed in a freeze dryer for 48 h. 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 amounts of petroleum ether and ethyl acetate, as well as any moisture present in the organic solvents, were further removed. The purified extract of phellodendri chinensis was subjected to a Propionibacterium acnes inhibition test. The petroleum ether was in a viscous liquid state after drying and could not be dissolved using water, so the extraction rate and minimum inhibitory concentration experimental results could not be obtained. The results are shown in Table 2 below. The purified water layer yellow powder had an inhibitory concentration of 1.56 mg / mL.

[0064] Table 2: Inhibition test results of the purified extract of phellodendri chinensis

[0065]

[0066] In some embodiments, a time-kill curve assay was performed. Propionibacterium acnes in the logarithmic growth phase was washed three times with phosphate buffer solution, and the turbidity tube was adjusted to 0.5 McFarland turbidity. The extract of phellodendri chinensis at 0 MIC, 1 MIC, 2 MIC, and 4 MIC was added to an equal volume of bacterial solution. Every 2 h, 100 μL of the sample was taken, gradient-diluted on an LB broth plate solid medium, and cultured in an anaerobic environment at 37°C for 48 h.

[0067] The extract of phellodendri chinensis was cultured with Propionibacterium acnes for 24 h to determine the time-kill curve. The colony count of the 0 MIC group did not change significantly after 24 h of culture. Compared with 0 MIC, the addition of 1 MIC and 2 MIC of the extract of phellodendri chinensis significantly reduced the colony count at 12 h of culture. The extract of phellodendri chinensis at 4 MIC significantly reduced the colony count at 2 h. Both low and high concentrations of the extract of phellodendri chinensis could significantly inhibit the growth of Propionibacterium acnes, and this was dose-dependent.

[0068] In some embodiments, the alkaline phosphatase activity assay is performed, the time-kill curve experiment is performed, the P. acnes in logarithmic growth phase is washed with phosphate buffer for 3 times, the turbidity tube is used to adjust to 0.5 Mclntock turbidity, the P. acnes is cultured with the 0 MIC group and 1 MIC group of the cortex phellodendri alcohol extract under anaerobic conditions at 37°C for 10 hours, samples are taken at 0, 2, 4, 6, 8, 10 hours, centrifuged at 8000g for 10 minutes, the supernatant is collected, and the alkaline phosphatase activity is determined by using an alkaline phosphatase detection kit (Nanjing Jiancheng Biological Institute). The alkaline phosphatase activity of the P. acnes in the 0 MIC group has little change after 10 hours of culture, compared with the 0 MIC group, the alkaline phosphatase activity of the P. acnes added with the cortex phellodendri alcohol extract is significantly increased, and significantly increases with the increase of the concentration of the cortex phellodendri alcohol extract.

[0069] The determination of the effect of the cortex phellodendri alcohol extract on the permeability of P. acnes is performed, the P. acnes in logarithmic growth phase is collected, washed with phosphate buffer for 3 times, and then suspended in phosphate buffer, and the bacterial suspension is adjusted to 0.5 Mclntock turbidity. The P. acnes is cultured with the 0 MIC group and 1 MIC group of the cortex phellodendri alcohol extract under anaerobic conditions at 37°C for 10 hours. Samples are taken at 0, 2, 4, 6, 8, 10 hours, and the leakage of nucleic acid and protein is determined by using a UV spectrophotometer at 260 nm and 280 nm. 2+ The content determination operation steps are the same as those of nucleic acid and protein, and the content of Ga 2+ ions is detected by using a Ga 2+ ion detection kit.

[0070] Nucleic acid and protein have maximum absorption peaks at 260 nm and 280 nm, respectively, and the absorption intensity is proportional to the concentration. The concentration of nucleic acid and protein in the extracellular fluid of P. acnes is measured by using the UV absorption method. After the P. acnes is cultured with the cortex phellodendri alcohol extract at various concentrations for 10 hours, the concentration of nucleic acid and protein is significantly increased at the 2nd hour, and significantly increases with the increase of the concentration of the cortex phellodendri alcohol extract.

[0071] Ga 2+ ions are also present in the cell membrane, so the leakage of Ga 2+ ions also indicates that the cell membrane of the bacteria is damaged. The change in the concentration of Ga 2+ ions in the 0 MIC group is not obvious. After the P. acnes is cultured with the cortex phellodendri alcohol extract for 10 hours, the concentration of Ca 2+ ions is significantly increased at the 2nd hour, and then tends to be stable, so the cortex phellodendri alcohol extract can destroy the cell wall and cell membrane of P. acnes to kill the bacteria.

[0072] In some embodiments, 8-week-old male ICR mice (body weight 30-32 g) were purchased from Liaoning Changsheng Experimental Animal Technology Co., Ltd. After one week of adaptive feeding, the mice were randomly divided into 6 groups: blank control group (CON), model group (MOD), adapalene group (Ada), low-dose group (1 MIC), medium-dose group (2 MIC) and high-dose group (4 MIC). The recovery of mice in each administration group was recorded by photography. Compared with the CON group, after injection of P. acnes, the mice in the MOD group developed cysts, papules on the abdominal surface, accompanied by ulceration and scabbing. The Ada group, 1 MIC, 2 MIC and 4 MIC groups had similar symptoms before treatment as the MOD group. After 1 week of Ada treatment, the symptoms did not change significantly, similar to the MOD group. After 5 days of treatment with Cortex Phellodendri ethanol extract solution, the skin lesions on the abdomen of mice in each group improved to varying degrees, among which the 2 MIC and 4 MIC groups had more obvious relief of abdominal inflammation, with most of the abscesses subsiding, and the 1 MIC group had slightly less inflammation and abscesses, which partially subsided compared to before treatment.

[0073] For the HE staining results of the mouse abdominal skin, the CON group had a rich number of skin appendages such as hair follicles and sebaceous glands in the dermis layer, with occasional lymphocytes; no obvious hyperkeratosis or other abnormalities were observed. Compared with the CON group, the MOD group had unclear boundaries between the dermis and subcutaneous connective tissue, a large amount of connective tissue hyperplasia, neovascularization, and a large number of lymphocyte and granulocyte infiltration, with swollen cells and loose cytoplasm; compared with the MOD group, the connective tissue and lymphocytes in each administration group were reduced, among which the 4 MIC group had more significant recovery of abdominal inflammation in mice after treatment.

[0074] Western blot method was used to detect the expression of related proteins in skin tissue, and it was found that Cortex Phellodendri ethanol extract could inhibit the secretion of cortex by down-regulating Src, Akt and mTOR proteins, thereby achieving the purpose of treating acne. Cortex Phellodendri ethanol extract can treat acne by inhibiting the Src / Akt / mTor pathway; the expression levels of Src, Akt and mTor proteins in the CON group were lower than those in the other groups; compared with the CON group, the expression levels of Src, Akt and mTor proteins in the MOD group were significantly increased; compared with the MOD group, the expression levels of Src, Akt and mTor in the skin tissue of each administration group were reduced to varying degrees.

[0075] Cortex Phellodendri ethanol extract can destroy the cell wall and cell membrane of P. acnes to kill the bacteria. The results of the time-killing curve showed that low and high concentrations of Cortex Phellodendri ethanol extract could significantly inhibit the growth of P. acnes in a dose-dependent manner, and Western blot results showed that Cortex Phellodendri ethanol extract could inhibit the secretion of cortex by down-regulating Src, Akt and mTOR proteins, thereby achieving the purpose of treating acne.

[0076] In some embodiments, the content of berberine in the Cortex Phellodendri alcohol extract is determined, and the average content of berberine is 15%.

[0077] In some embodiments, by combining mass spectrometry total ion chromatogram and ultraviolet absorption chromatogram, 7 compounds are found in the transdermal skin sample as shown in Table 3.

[0078] Table 3: Cortex Phellodendri alcohol extract transdermal skin ingredient table

[0079]

[0080] In some embodiments, the Cortex Phellodendri alcohol extract is used to prepare a coating agent. Appropriate amounts of PVA 22-88, PVA 35-80, and distilled water are added; appropriate amounts of PVA 574 and PVA L-508w and 50% ethanol are added, and all are dissolved to a concentration of 10%. Single factor experiments are used to investigate the effects of adding different concentrations of glycerol, Tween-80, and different drug amounts on tensile strength and elongation rate, so as to screen the appropriate prescription range. Single factor analysis is shown in Table 4.

[0081] Table 4: Single factor analysis table

[0082]

[0083] On the basis of the single factor experiment, glycerol, Tween-80, and drug loading are used as factors, and tensile strength, elongation rate, and film forming time are used as indexes. SPSS is used for orthogonal test design. The orthogonal test design is shown in Table 5.

[0084] Table 5: Orthogonal test design table

[0085]

[0086] In some embodiments, the PVA with 5% glycerol is the initial formula, and glycerol is added at gradients of 5%, 10%, 15%, 20%, and 25% to measure the elongation rate 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 film is too thin and soft, so the elongation rate and tensile strength cannot be measured. Compared with the initial formula, the elongation rate of PVA 22-88 and PVA 35-80 is the largest when the glycerol is 15%, and the tensile strength is the largest when the glycerol is 5%. The elongation rate of PVA 574 and PVA L-508W is the largest when the glycerol is 10%, and the tensile strength is the largest when the glycerol is 5%. The tensile strength of the four types of PVA decreases with the increase of the amount of glycerol. Considering both indexes, the mechanical strength of PVA 22-88 and PVA 35-80 is the best when the glycerol addition concentration is 15%, and the mechanical strength of PVA 574 and PVA L-508W is the best when the glycerol addition concentration is 10%.

[0087] In some embodiments, the PVA 22-88 and PVA 35-80 glycerol concentration 15% is set as a fixed value, the PVA 574 and PVA L-508W glycerol concentration 10% is set as a fixed value, and the Tween-80 is added in gradients of 0.5%, 1%, 1.5%, 2% and 2.5% to measure the elongation and mechanical properties. After adding Tween-80, the elongation of the remaining 3 PVAs significantly decreased after adding 0.5% Tween-80, except for PVA 35-80. Compared with the addition of 0.5% Tween-80, the elongation of PVA 22-88 significantly increased after the addition of 2.5% Tween-80. The addition of Tween-80 had no significant effect on the tensile strength of PVA. In summary, the Tween-80 addition concentration of PVA 22-88, PVA 35-80 and PVA L-508W is 2%, and the Tween-80 addition concentration of PVA 574 is 1%, at which the mechanical properties of the four PVAs are most similar.

[0088] Different glycerol, Tween-80 concentrations and drug loading have different effects on the mechanical properties of PVA, and the glycerol concentration and drug loading have a more significant effect on the elongation, while the Tween-80 has a smaller effect. The four types of PVA can form a coating agent under experimental conditions, among which PVA 22-88 has a greater tensile strength and better elongation than other PVAs, and each experimental factor has a more significant effect on it, so PVA 22-88 is selected as the subsequent drug-loaded matrix.

[0089] An orthogonal test was conducted on different glycerol, Tween-80 concentrations and drug loading, and the test results are shown in Table 6. Based on the orthogonal test results and range analysis, the prescription of the coating agent of Phellodendri Chinensis Ammonia Extract was screened through the orthogonal test, and three prescriptions were determined based on the elongation, tensile strength and film forming time as indicators:

[0090] Prescription 1: PVA 22-88 mass ratio 10%, glycerol mass ratio 10%, Tween mass ratio 2.5%, and Phellodendri Chinensis Ammonia Extract mass ratio 6%;

[0091] Prescription 2: PVA 22-88 mass ratio 10%, glycerol mass ratio 20%, Tween mass ratio 1.5%, and Phellodendri Chinensis Ammonia Extract mass ratio 6%;

[0092] Prescription 3: PVA 22-88 mass ratio 10%, glycerol mass ratio 10%, Tween mass ratio 1.5%, and Phellodendri Chinensis Ammonia Extract mass ratio 2%.

[0093] Table 6: Orthogonal test results of glycerol, Tween-80 concentration, PVA and drug loading

[0094]

[0095] In some embodiments, the preparation steps of the Phellodendri Chinensis Alcohol Extract film coating agent include,

[0096] P1 : PVA 22-88 is added to distilled water and stirred until completely dissolved, after dissolution the PVA 22-88 is at a concentration of 10%;

[0097] P2: After cooling to 40-50°C, glycerol, Tween-80 and Phellodendri Chinensis Alcohol Extract are added in sequence and stirred uniformly;

[0098] P3: After standing and defoaming, the film is coated in a mold, dried for 15-30 min, and a film with a thickness of 0.1-0.3 mm is formed.

[0099] In some embodiments, rabbit skin is installed on a vertical diffusion cell (diffusion area = 1.77 cm 2 ), 0.2 ml of Phellodendri Chinensis Alcohol Extract film coating agent is evenly applied to the stratum corneum of the skin, the diffusion cell contains 4 mL of phosphate buffered saline receiving solution, and the stirring speed is 600 rpm. The temperature is maintained at 32±0.5°C. Samples are taken at 2, 4, 6, 8, 12 and 24 h, respectively, and new receiving solution is immediately added to maintain the balance of the cell. The samples are analyzed by HPLC method. All in vitro experiments are repeated 3 times, and the cumulative curve of the cumulative permeation amount Q per unit area of the drug with time t is drawn.

[0100] The chromatographic column is Welch Ultimate® LP-C18 (4.6 x 250 mm, 5 μm), HPLC chromatography is performed, the mobile phase is A acetonitrile and B 0.1% phosphoric acid solution, the detection wavelength is 345 nm, the column temperature is 30°C, the flow rate is 1 mL / min, and the gradient elution program is shown in Table 7:

[0101] Table 7: Transdermal chromatographic condition elution program table

[0102]

[0103] The in vitro transdermal parameter calculation formula is as follows:

[0104]

[0105] In the formula: 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.

[0106] According to the tensile strength, elongation and film forming time, three prescriptions are selected, and the final selection is made by in vitro transdermal results. The transdermal parameter calculation results are shown in Table 8. By comparison, prescription 1 is selected as the best prescription for subsequent efficacy tests.

[0107] Table 8: Calculated results of the percutaneous parameters of the Cortex Phellodendri alcohol extract

[0108]

[0109] Therefore, in the present application, the main components of the coating agent are Cortex Phellodendri alcohol extract 2-6%, PVA 22-88 8-12%, glycerol 10-20%, Tween-80 1.5-2.5%, and the rest is water, and when the mass percentage of PVA 22-88 is 10%, the mass percentage of glycerol is 10%, the mass percentage of Tween is 2.5%, and the mass percentage of Cortex Phellodendri alcohol extract is 6%, the best percutaneous experimental effect is achieved.

[0110] In some embodiments, the pharmacodynamic test shows that after 5 days of continuous administration, the mice in the Cortex Phellodendri alcohol extract coating agent group have basically recovered on the 4th day without using any penetration enhancer, and the model group still has cysts, indicating that the coating agent has the effect of treating acne.

[0111] The above is a further detailed description of the present application in combination with specific preferred embodiments. For those of ordinary skill in the art to which the present application belongs, without departing from the present application, a number of simple deductions or substitutions can be made, which should be considered to fall within the scope of patent protection determined by the claims submitted.

Claims

1. A film-coating agent prepared from a phellodendron amurense Rupr. alcohol extract, characterized by: The coating film agent comprises a Cortex Phellodendri 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; The mass percentage components of the coating film agent are as follows: Cortex Phellodendri alcohol extract 2-6%, PVA22-88 8-12%, glycerol 10-20%, Tween-80 1.5-2.5%, and the rest is water. The Cortex Phellodendri alcohol extraction method comprises the following steps: S1: Cortex Phellodendri is weighed and soaked in an ethanol solution, and then extracted by water bath reflux, for 2 hours the first time, and the filtrate is collected; S2: The residue is added with ethanol solution and extracted for 1.5 hours again, and the filtrates of the two times are mixed; S3: The mixed filtrate is concentrated by using a rotary evaporator, centrifuged in a centrifuge at a speed of 4000 rpm for 5 minutes, and then placed in a 4℃ sterile environment for overnight; S4: The supernatant after centrifugation is collected, frozen in a-20℃ refrigerator, and then transferred to a freeze dryer for freeze-drying for 48 hours to form a powder; S5: The powder is taken and diluted with water, poured into a separatory funnel, and then petroleum ether is added and oscillated for extraction, and then left to stand; S6: The upper and lower layer solutions are collected respectively, and after repeated for 3 times, ethyl acetate is added for extraction; S7: The upper and lower layer solutions after extraction are collected and repeated for 3 times, the water layer is frozen in-20℃ for 8 hours, and then placed in a freeze dryer for freeze-drying for 48 hours to form a powder, thereby obtaining the Cortex Phellodendri alcohol extract.

2. The coating agent prepared from the Phellodendri Chinensis Cortex extract according to claim 1, characterized by: In S1, the volume fraction of ethanol is 70%, and the water bath temperature is 70℃.

3. The coating agent prepared from the Phellodendri Chinensis Cortex extract according to claim 1, characterized by: The coating film agent is used for in-vitro transdermal experiment on rabbit skin, and the in-vitro transdermal parameter calculation formula of the coating film agent is as follows: ， In the formula, Q is the cumulative penetration per unit area, F is the drug release rate, J is the penetration 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.

4. The film-coating agent prepared from the Phellodendri Chinensis Cortex extract according to claim 1, characterized by: When the mass percentage of PVA22-88 in the coating film agent is 10%, the mass percentage of glycerol is 10%, the mass percentage of Tween-80 is 2.5%, and the mass percentage of Cortex Phellodendri alcohol extract is 6%, the cumulative penetration per unit area of the coating film agent is 82.56±10.45μg / cm2 / h after in-vitro transdermal experiment for 24 hours.

5. The film-coating agent prepared from the Phellodendri Chinensis Cortex extract according to any one of claims 1 to 4, characterized by: The Cortex Phellodendri alcohol extract comprises Phellodendrine, 3-O-feruloylquinic acid, 5-O-feruloylquinic acid and Berberine.

6. Use of the coating agent prepared from the extract of Phellodendri Cortex according to claim 1 in the preparation of an anti-acne medicament, characterized in that: The coating film agent treats acne by inhibiting the Src / Akt / mTOR signal pathway of Propionibacterium acnes, which is specifically manifested as follows: the coating film agent reduces the expression levels of Src, Akt and mTOR proteins in skin tissue, and destroys the integrity of cell walls and cell membranes of Propionibacterium acnes, thereby causing leakage of alkaline phosphatase, nucleic acid and proteins of Propionibacterium acnes.

7. The method for preparing a coating agent prepared from the extract of Phellodendri Cortex according to claim 1, characterized in that: The method comprises the following steps: P1: PVA22-88 is added to distilled water and stirred until completely dissolved, and the concentration of PVA22-88 after dissolution is 10%; P2: After cooling to 40-50℃, glycerol, Tween-80 and Cortex Phellodendri alcohol extract are added in sequence and stirred uniformly; P3: After standing and defoaming, coat in the mold, dry for 15-30 min, to form a film with a thickness of 0.1-0.3 mm.

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