Product for treating acne and preparation method thereof

Abstract

The invention relates to the technical field of medicines, in particular to a product for treating acne and a preparation method thereof. According to the invention, firstly, asiaticoside and beta-cyclodextrin are subjected to inclusion so as to improve the solubility and bioavailability of asiaticoside; and adding the asiaticoside clathrate compound into a chitosan system to form a product for treating acne. The product for treating acne can effectively improve the solubility and bioavailability of asiaticoside and enhance the effect of asiaticoside in acne treatment, has the potential to become a new safe and effective skin disease treatment method, and provides a new thought and scientific basis for future skin disease treatment.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and particularly to a product for treating acne and a preparation method thereof. Background Art

[0002] Acne is a common skin disease that particularly affects adolescents and young populations, causing significant impacts on the physical health and psychological state of patients. Traditional acne treatment methods mainly include topical application of antibiotics, vitamin A derivatives, etc. However, these therapies are often accompanied by a series of adverse reactions, such as skin irritation, dryness, and antibiotic resistance. These limitations have prompted researchers to explore alternative treatment means that are safer, more effective, and can improve patient compliance. Summary of the Invention

[0003] Based on the above, the present invention provides a product for treating acne and a preparation method thereof.

[0004] To achieve the above object, the present invention provides the following solutions:

[0005] One of the technical solutions of the present invention is a preparation method of a product for treating acne, comprising the following steps:

[0006] Add the asiaticoside solution to the β-cyclodextrin solution, stir, then rotary evaporate to remove the organic solvent, and then freeze-dry to obtain asiaticoside inclusion complex;

[0007] Dissolve chitosan in acetic acid solution, then add polyvinyl alcohol to dissolve, and then add glutaraldehyde for crosslinking reaction to obtain a crosslinked solution;

[0008] Add the asiaticoside inclusion complex, hydroxypropyl methylcellulose, ethylparaben, and water to the crosslinked solution to obtain a product for treating acne (i.e., asiaticoside chitosan composite nanogel).

[0009] In the present invention, hydroxypropyl methylcellulose (HPMC), as an ideal hydrogel matrix material, forms a covalent bond network through reaction with the crosslinking agent glutaraldehyde. Hydroxypropyl methylcellulose is suitable for scenarios requiring thermosensitivity, biocompatibility, and sustained release function. The present invention also separately experimented with replacing hydroxypropyl methylcellulose with gelatin, carbomer, chitosan, hyaluronic acid, methylcellulose, sodium carboxymethylcellulose, etc. as the hydrogel matrix material. The results showed that only when hydroxypropyl methylcellulose was used, the asiaticoside chitosan composite nanogel prepared had the best treatment effect on acne.

[0010] In the present invention, ethylparaben plays an antiseptic role. Other commonly used preservatives in the art, such as sodium benzoate, sorbic acid, ethylparaben, benzalkonium bromide, etc., are also applicable to the present invention.

[0011] In a preferred embodiment of the present invention, the asiaticoside solution is prepared by dissolving asiaticoside in absolute ethanol; the β-cyclodextrin solution is prepared by dissolving β-cyclodextrin in water; the mass ratio of asiaticoside in the asiaticoside solution to β-cyclodextrin in the β-cyclodextrin solution is 1:(3 - 5), preferably 1:4.

[0012] In a preferred embodiment of the present invention, the temperature of the stirring is room temperature, and the time is 15 - 17 h, preferably 16 h.

[0013] The present invention does not make special limitations on the parameter settings of freeze-drying, and the freeze-drying parameter settings (vacuum degree, cold trap temperature, time) well-known to those skilled in the art can be adopted. For example, the vacuum degree is 10 Pa, the cold trap temperature is -56 °C, and the time is 24 h.

[0014] In a preferred embodiment of the present invention, the mass concentration of the acetic acid solution is 0.1%; the concentration of chitosan in the cross-linking solution is 8 mg / mL; the concentration of polyvinyl alcohol in the cross-linking solution is 30 mg / mL; the volume-mass ratio of glutaraldehyde to chitosan is (80 - 120) μL:0.8 g, preferably 100 μL:0.8 g.

[0015] In a preferred embodiment of the present invention, the mass ratio of asiaticoside in the asiaticoside solution to chitosan is 5:8.

[0016] In a preferred embodiment of the present invention, the temperature for dissolving polyvinyl alcohol is set at 80 °C. After the polyvinyl alcohol is dissolved, the temperature is lowered to room temperature and then glutaraldehyde is added for cross-linking reaction; the cross-linking reaction is specifically carried out at room temperature for 25 - 35 min, preferably 30 min.

[0017] In a preferred embodiment of the present invention, the concentration of chitosan in the product is 8 mg / mL.

[0018] In a preferred embodiment of the present invention, the concentration of hydroxypropyl methylcellulose in the product is 5 mg / mL; the concentration of ethylparaben in the product is 0.5 mg / mL.

[0019] When preparing a product for treating acne, the temperature of the stirring is room temperature, and the time is 1 h.

[0020] The second technical solution of the present invention is a product prepared by the above preparation method.

[0021] The third technical solution of the present invention is the application of the above product in the preparation of a drug for treating acne.

[0022] The present invention discloses the following technical effects:

[0023] The present invention provides a preparation method for a product (centella asiatica total glycoside chitosan composite nanogel) for treating acne. The centella asiatica total glycoside chitosan composite nanogel prepared by using this method exhibits good stability, an appropriate pH value, and good spreadability. The results of animal experiments show that, compared with the model group, in the process of acne treatment in mice smeared with the centella asiatica total glycoside chitosan composite nanogel, the inflammation is significantly reduced and the skin damage is significantly repaired. Therefore, the centella asiatica total glycoside chitosan composite nanogel of the present invention can effectively improve the solubility and bioavailability of centella asiatica total glycoside, enhance its effect in acne treatment, and has the potential to become a new safe and effective skin disease treatment method, providing new ideas and scientific basis for future skin disease treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0025] Figure 1 It is a high performance liquid chromatography (HPLC) chromatogram for the specificity test; wherein, A is the negative sample solution, B is the reference substance solution, and C is the test sample solution (centella asiatica total glycoside chitosan composite nanogel solution).

[0026] Figure 2 It is the skin morphology of mice in each group after 7 days of treatment.

[0027] Figure 3 It is the pathological picture of the back skin tissue of mice in each group after 7 days of treatment (scale bar: 200 μm).

[0028] Figure 4 It is the contents of IL-6 (A), IL-β (B), and TNF-α (C) in the sera of mice in each group after 7 days of treatment (*P < 0.05, **P < 0.01).

[0029] Figure 5 It is the state diagram of the centella asiatica total glycoside chitosan composite nanogel. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0030] Now, various exemplary embodiments of the present invention will be described in detail. This detailed description should not be construed as a limitation on the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0031] It should be understood that the terms used in the present invention are only for describing specific embodiments and are not intended to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0032] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0033] Without departing from the scope or spirit of the present invention, various improvements and modifications can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the present invention specification are obvious to those skilled in the art. The present invention specification and examples are merely exemplary.

[0034] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0035] The "%" mentioned in the present invention represents mass percentage unless otherwise specified.

[0036] The "room temperature" mentioned in the present invention represents 20 - 30 °C unless otherwise specified.

[0037] Asiaticoside is an active compound derived from the Chinese herbal medicine Centella asiatica. Due to its significant anti-inflammatory, antibacterial, and tissue-healing-promoting effects, it shows important potential in the treatment of inflammatory skin diseases including acne. However, the application of asiaticoside is limited by its poor water solubility and low bioavailability. To address this problem, researchers have begun to focus on how to improve the solubility and bioavailability of asiaticoside.

[0038] β-Cyclodextrin is a cyclic molecule composed of 7 D-glucose units linked by α-1,4-glycosidic bonds. Its unique "hydrophilic outer surface - hydrophobic inner cavity" structure endows it with excellent solubilization ability. β-Cyclodextrin can form stable inclusion complexes with hydrophobic drug molecules through inclusion, thus significantly improving the solubility of poorly soluble drugs. In addition, due to its good biocompatibility, biodegradability and unique film-forming properties, chitosan is widely used in the fields of drug delivery and tissue engineering. Chitosan can not only protect the active ingredients of drugs, but also significantly enhance the therapeutic effect by increasing the residence time of drugs on the skin surface and improving the transdermal absorption rate. As a novel drug carrier, nanogel has a high water content and good adhesiveness, which is very suitable for skin drug delivery. The small size of nanogel enables it to penetrate more easily into different layers of the skin, thereby increasing the local drug concentration and enhancing the therapeutic effect. Therefore, preparing asiaticoside into an inclusion complex with β-cyclodextrin can not only solve the problem of its low solubility, but also further improve its therapeutic performance through the synergistic effect with chitosan.

[0039] The present invention utilizes the synergistic and enhancing properties of β-cyclodextrin and chitosan to prepare asiaticoside chitosan composite nanogel (i.e., a product for treating acne), and evaluates its quality. On this basis, a mouse acne model is constructed by using Propionibacterium acnes to model mice. Subsequently, the composite nanogel is used for topical treatment to study its therapeutic effect on the acne model mice. The present invention not only opens up a new direction for the application of asiaticoside, but also provides a scientific basis for the future development of safe and efficient skin disease treatment methods.

[0040] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art. The reagents or raw materials used, unless otherwise specified, are all purchased from commercial channels or have been made public.

[0041] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0042] Example 1

[0043] 1 Instruments and Reagents

[0044] 1.1 Instruments

[0045] LC-20AD high performance liquid chromatograph (photodiode array detector PDA, Shimadzu Corporation, Japan); Cary-300 ultraviolet-visible spectrophotometer (Agilent Technologies, Inc., USA); Electrothermal constant temperature forced air drying oven (Nantong Hunan Scientific Instrument Co., Ltd.); Digital display constant temperature water bath (Changzhou Hongze Experimental Technology Co., Ltd.); One over one hundred thousandth electronic analytical balance (Sartorius Instruments Co., Ltd., Beijing); Centrifuge (Thermo Fisher Scientific Inc.); pH meter (Shanghai INESA Scientific Instrument Co., Ltd.).

[0046] 1.2 Reagents

[0047] Madecassoside was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; β-cyclodextrin was purchased from Shanghai Bide Pharmaceutical Technology Co., Ltd.; Chitosan (molecular weight 150KDa) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; Polyvinyl alcohol (molecular weight 31-50KDa) was purchased from Merck Chemical Reagent Co., Ltd.; Propionibacterium acnes was purchased from Ningbo Mingzhou Biotechnology Co., Ltd.; Tumor necrosis factor-α (TNF-α), interleukin 10 (IL-10), interleukin 1β (IL-1β) detection kits were purchased from Shanghai Enzyme-linked Biotechnology Co., Ltd.

[0048] 1.3 Animals

[0049] 32 SPF-grade 8-week-old ICR mice were purchased from the Animal Experiment Center of Nantong University. They were fed with standard mouse feed conventionally and allowed to eat and drink freely in the same environment.

[0050] 2 Methods and Results

[0051] 2.1 Determination of Madecassoside Content

[0052] 2.1.1 Chromatographic Conditions

[0053] Chromatographic column: Agilent 5HC C(18)2 (250mm×4.6mm, 5μm), mobile phase: 2mM β-cyclodextrin solution–acetonitrile = 72:28, running for 25 min; Flow rate: 1 mL / min; Detection wavelength: 205 nm; Column temperature: 40 °C; Injection volume: 10 μL.

[0054] 2.1.2 Preparation of Solutions

[0055] Reference solution: Take 50 mg of madecassoside reference substance, place it in a 50 mL volumetric flask, then add an appropriate amount of methanol, dissolve it by ultrasonic wave, and then dilute it to the scale with methanol and shake well to obtain the madecassoside reference stock solution; Accurately measure 8 mL of this stock solution and place it in a 20 mL volumetric flask, dilute it to the scale with methanol and shake well.

[0056] Test solution: Accurately weigh 1600 mg of asiaticoside chitosan composite nanogel (prepared according to "2.2"), place it in a 10 mL volumetric flask, dissolve it ultrasonically with 50% methanol solution, centrifuge and take 5 mL of the supernatant, place it in a 10 mL volumetric flask, and dilute it to the mark with methanol.

[0057] Negative sample solution: According to the prescription ratio and preparation process of asiaticoside chitosan composite nanogel, first prepare a negative sample without asiaticoside. Then, prepare the negative sample solution according to the method specified under the item of "Test solution".

[0058] 2.1.3 Method validation

[0059] (1) Specificity test

[0060] Precisely pipette 10 μL each of the negative sample solution, test solution, and reference solution under "2.1.2". Inject and detect according to the chromatographic conditions under "2.1.1", and record the chromatogram. The chromatogram is shown in Figure 1 . The result shows that the elution time of asiaticoside is 12.560 min. The separation between asiaticoside and the adjacent peak is 16.16 (>1.5). This indicates that other components in the asiaticoside chitosan composite nanogel do not interfere with the detection of the main component, and the method has good specificity.

[0061] (2) Linearity and range

[0062] Taking the concentration of asiaticoside (0.4 mg / mL) as 100%, investigate the linear relationship in the concentration range of 50% - 150%. Precisely pipette 2, 2.4, 3.2, 4, 4.8, 6 mL of the reference stock solution under "2.1.2" into 10 mL volumetric flasks respectively, and dilute them to the mark with methanol. Take 10 μL of each concentration solution, inject and analyze according to the chromatographic conditions under "2.1.1", and record the chromatogram. Taking the peak area (Y) as the ordinate and the concentration (X, mg / mL) as the abscissa, perform linear regression. The result shows that the linear regression equation is y = 2388.4X + 8.2757, R2 = 0.9999, and asiaticoside has a good linear relationship in the range of 0.209 - 0.626 mg / mL.

[0063] (3) Precision test

[0064] Take the reference solution under "2.1.2", inject and detect according to the chromatographic conditions specified under "2.1.1". Perform 6 consecutive determinations, and record the chromatogram each time. The result shows that after 6 consecutive injections, the relative standard deviation (RSD) of the asiaticoside peak area is 0.65% (<2%), indicating good instrument precision.

[0065] (4) Repeatability test

[0066] Take the same batch of asiaticoside chitosan composite nanogel (prepared according to "2.2"), and prepare 2 portions of reference solution and 6 portions of test solution respectively according to the method under "2.1.2". Inject and detect according to the chromatographic conditions under "2.1.1", and record the chromatogram. The external standard method is adopted, and the content and RSD value of asiaticoside in the asiaticoside chitosan composite nanogel are calculated according to the peak area. The results show that the RSD of the content of the 6 portions of test solution is 0.57% (<2%), indicating that the repeatability of this method is good.

[0067] (5) Solution stability test

[0068] Take the same batch of asiaticoside chitosan composite gel solution (prepared according to "2.2"), and prepare the test solution according to the method under "2.1.2". Place this solution at room temperature, sample and detect at different time points within 24 hours, and record the chromatograms at each time point. The results show that the RSD of the peak area of asiaticoside is 0.85%, less than 2%, indicating that the test solution is basically stable when placed at room temperature for 24 h.

[0069] (6) Recovery test

[0070] Accurately weigh appropriate amounts of asiaticoside at 80%, 100%, and 120% of the prescription amount (the mass of asiaticoside recorded in 2.2, 0.5 g), add them to the negative sample solution described under "2.1.2" to simulate the preparation of asiaticoside chitosan composite nanogel. Then, prepare the test solution according to the method under "2.1.2", and prepare 3 portions for each concentration. Prepare another 2 portions of asiaticoside reference solution. Accurately measure 10 μL of each of the test solution and the reference solution respectively, inject and detect according to the chromatographic conditions under "2.1.1", and record the chromatogram. By the external standard method, calculate the content of asiaticoside in the gel according to the peak area, and calculate the recovery rate and RSD value. The results show that the average recovery rate of asiaticoside is 98.95%, and the RSD is 1.25%, indicating that this method has good accuracy.

[0071] 2.2 Preparation of asiaticoside chitosan composite nanogel (abbreviation: asiaticoside gel)

[0072] Preparation of asiaticoside inclusion complex: Prepared by the "saturated aqueous solution method". Accurately weigh 2 g of β-cyclodextrin, place it in a 250 mL flask, measure 100 mL of ultrapure water, and ultrasonically dissolve until completely dissolved. Another accurately weigh 0.5 g of asiaticoside, ultrasonically dissolve it in 250 mL of absolute ethanol, and slowly add it to the β-cyclodextrin solution, stir for 16 h, concentrate and remove the organic solvent with a rotary evaporator, and freeze-dry the solution to obtain asiaticoside inclusion complex.

[0073] Crosslinking of chitosan and glutaraldehyde: Weigh 0.8 g of chitosan, and fully swell and dissolve it with 100 mL of (0.1% acetic acid) solution. Add 3 g of polyvinyl alcohol to it, and continuously stir at 80 °C until the polyvinyl alcohol is completely dissolved, then slowly cool to room temperature. Then, under continuous stirring, dropwise add 100 μL of 50% glutaraldehyde aqueous solution, and allow it to fully undergo crosslinking reaction for 30 min.

[0074] Preparation of gel: Under ice bath conditions, add the prepared asiaticoside inclusion complex, 0.5 g of hydroxypropyl methylcellulose (HPMC), and 0.05 g of ethylparaben to the crosslinked solution of chitosan and glutaraldehyde, and make up to 100 mL with water. Continue to stir for 1 h to make it evenly mixed to obtain the gel.

[0075] 2.3 Quality evaluation of asiaticoside chitosan composite nanogel

[0076] 2.3.1 Appearance

[0077] This product is a light yellow semi-transparent gel, without bubbles on the surface, and has good spreadability.

[0078] 2.3.2 pH inspection

[0079] Perform pH detection on the asiaticoside chitosan composite nanogel, and the result shows that the pH is 6.5, which is similar to the pH of human skin surface.

[0080] 2.3.2 Stability evaluation

[0081] Perform stability determination on the asiaticoside chitosan composite nanogel, mainly including centrifugation test, heat resistance test, and cold resistance test.

[0082] Centrifugation test: Centrifuge the asiaticoside chitosan composite nanogel at a speed of 3000 r / min for 20 minutes and then take samples for observation. Observe whether the appearance of the gel is uniform, and whether there are phenomena such as stratification and precipitation.

[0083] Heat resistance test: Place the asiaticoside chitosan composite nanogel in an oven at 60 °C, take it out after 2 h, and observe after it returns to room temperature. Observe whether there are phenomena such as stratification and precipitation in the gel.

[0084] Cold resistance test: Place the asiaticoside chitosan composite nanogel in a 4 °C refrigerator, and take samples for observation on day 0, day 5, and day 10 respectively. Observe the appearance properties, uniformity, spreadability, and stability of the gel.

[0085] The results show that: After the centrifugation test, heat resistance test, and cold resistance test, the asiaticoside chitosan composite nanogel did not show phenomena such as stratification and precipitation, and had good uniformity and spreadability.

[0086] 2.4 Preliminary pharmacodynamic study

[0087] 2.4.1 Establishment and Intervention Method of Acne Mouse Model

[0088] Propionibacterium acnes (P. acnes) was inoculated into brain heart infusion medium. After subculturing and activating at 37°C, it was anaerobically cultured and proliferated to a bacterial solution of 1×10 9 CFU / mL for subsequent experiments. SPF-grade ICR mice were randomly divided into four groups: normal control group, model group, adapalene gel group, and asiaticoside gel group, with 8 mice in each group. During the experiment, all mice were anesthetized by intraperitoneal injection of 5% chloral hydrate (dose: 0.006 mL / g). Subsequently, hair removal was performed on the backs of the mice. An acne mouse model was constructed: mice in the normal control group were injected with 50 μL of phosphate buffer solution (PBS) on the back, while the other three groups were continuously injected with 50 μL of the prepared P. acnes bacterial solution on the back for 5 days. The criteria for successful modeling were: raised bumps appeared on the backs of the mice, the tops of the bumps were white, the bases were red, and significant inflammatory reactions such as swelling, inflammation, etc. occurred.

[0089] After successful model establishment, the mice in the model group continued to be maintained under normal feeding conditions. At the same time, the mice in the drug administration groups were required to evenly apply the corresponding gel on their backs every morning and evening. The gel application should fully cover the acne area of the mice, with a thickness of approximately 0.3 - 0.5 mm. This drug administration process lasted for 7 days. After the last drug administration, the mice were fasted (with water supply maintained) for 24 hours. Subsequently, blood samples were collected by orbital blood sampling and left to stand at room temperature until the blood coagulated. Then, the samples were placed at 4°C and centrifuged at a speed of 3000 revolutions per minute for 15 minutes to separate the serum, which was stored in a -80°C refrigerator for subsequent use. At the end of the experiment, the back skin tissues of the mice in each group were excised. Part of the skin tissue was fixed with 4% paraformaldehyde solution; the remaining skin tissue was stored in a -80°C refrigerator for subsequent analysis.

[0090] 2.4.2 Detection Indexes

[0091] (1) Observation of Mouse Back Skin

[0092] During the process of mouse modeling and drug intervention, the backs of the mice in each group were observed every day, paying special attention to phenomena such as swelling, bumps, and ulcers. The drug intervention treatment lasted for 7 days.

[0093] Results: On the fifth day of the experiment, it was observed that, except for the normal control group, lumps formed on the backs of the mice in the other groups, and these lumps subsequently ulcerated. The base of the lumps showed signs of redness and was accompanied by an inflammatory reaction, specifically manifested as redness and swelling symptoms. The above results indicate that a mouse acne model has been successfully constructed. After successful model establishment, the mice in the adapalene gel group and the asiaticoside gel group were respectively smeared with the corresponding drugs for 7 days, and the results are shown in Figure 2 , compared with the mice in the model group, the inflammatory redness and swelling on the backs of the mice in the drug administration groups were alleviated to varying degrees, and the small lumps on the backs began to heal.

[0094] (2) Observation of histopathological changes in mouse skin tissue by HE staining

[0095] The skin tissue samples fixed with 4% paraformaldehyde were dehydrated according to the standard procedure, and then embedded in paraffin for sectioning. After the obtained sections were stained with hematoxylin-eosin (HE), they were dehydrated through a series of gradient ethanol solutions, followed by transparency treatment with xylene, and finally sealed with neutral gum. After these steps were completed, the histopathological changes in the skin lesions of the backs of the mice in each group were observed and analyzed in detail using an optical microscope. Figure 3 The HE staining results of the mouse back skin tissue are shown. Compared with the normal control group, the skin of the mice in the model group showed a significant thickening of the epidermal layer, and the skin barrier function was damaged, specifically manifested as epidermal ulceration. In addition, fibrous tissue hyperplasia occurred in the dermis of the mice in the model group, and a large number of inflammatory cell infiltrations were visible. In contrast, in the experimental group of mice treated with adapalene gel or asiaticoside gel, the degree of epidermal thickening, the range of fibrous tissue hyperplasia, and the degree of inflammatory cell infiltration on the back skin were all reduced to varying degrees. This result indicates that the above two drugs have a positive effect on improving the skin pathological state of mice.

[0096] (3) Detection of the contents of IL-6, IL-1β, and TNF-α in mouse serum by enzyme-linked immunosorbent assay (ELISA)

[0097] To detect the expression levels of interleukin-6 (IL-6), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α) in mouse serum, we first thawed the serum samples and, according to the operation guide of the ELISA kit, took an appropriate amount of mouse serum for the following experimental steps: added a horseradish peroxidase-labeled detection antibody to the sample, and then incubated it at 37 °C for 60 minutes. After that, added the chromogenic agent and reacted for 15 minutes in the dark, and then immediately added the stop solution to terminate the reaction. The absorbance value of the sample was measured at a wavelength of 450 nm using an enzyme-labeled instrument, a standard curve was drawn based on the obtained data, and the concentrations of each inflammatory factor were calculated accordingly.

[0098] The results are shown inFigure 4 Compared with the normal group of mice, the levels of IL-6, IL-1β, and TNF-α in the model group of mice were significantly increased. Compared with the model group, the expression levels of these three inflammatory factors were significantly decreased in the groups of mice treated with adapalene gel or asiaticoside gel. It is worth noting that the levels of IL-6 and TNF-α inflammatory factors in the mice of the asiaticoside gel group were lower than those in the adapalene gel group, indicating that asiaticoside gel may have a more significant effect in inhibiting these inflammatory factors.

[0099] 2.5 Statistical analysis

[0100] The data were statistically analyzed using Graphpad 8.0 software, and the experimental data were expressed as mean ± standard deviation. One-way analysis of variance was used for comparison of differences between groups, and P < 0.05 was considered statistically significant.

[0101] Acne is a common skin disease, mainly manifested as skin lesions such as comedones, pustules, and papules on the face, back, chest, etc., usually accompanied by obvious inflammatory reactions. Its pathogenesis is very complex, involving the interaction of multiple factors, including excessive secretion of sebaceous glands, follicular occlusion, dysregulation of the skin microbiota, and abnormal immune responses. In recent years, more and more studies have found that inflammatory factors play a key role in the occurrence and development of acne, especially pro-inflammatory cytokines such as IL-6, IL-1β, and TNF-α, which are considered to be the main pathogenic factors in the inflammatory reaction of acne skin. Regulating the expression levels of these inflammatory factors and inhibiting the local inflammatory reaction of the skin have become one of the important directions for the treatment of acne.

[0102] During the occurrence of acne, pro-inflammatory cytokines such as IL-6, IL-1β, and TNF-α play a crucial role. IL-6 is a multifunctional cytokine that can further exacerbate the pathological changes of acne by promoting the hyperplasia and secretion of sebaceous glands and accelerating follicular keratinization. As a potent pro-inflammatory factor, IL-1β can activate immune cells in the skin, leading to local inflammatory reactions. TNF-α enhances the inflammatory reaction and exacerbates tissue damage by activating immune cells. Studies have shown that the expression of inflammatory factors is closely related to the clinical severity of acne. Therefore, inhibiting their excessive release is of great significance in reducing the inflammatory reaction of acne and improving clinical symptoms.

[0103] As a natural medicine, asiaticoside can reduce local inflammatory reactions by regulating the functions of immune cells and inhibiting the secretion of IL-6, IL-1β, and TNF-α. In addition, asiaticoside also has antioxidant effects, which can reduce the oxidative stress reaction caused by acne and provide a favorable environment for the healing and repair of the skin.

[0104] However, asiaticoside, as a compound with poor water solubility, has certain limitations in its clinical application. To improve its activity and overcome the problem of low solubility, researchers prepared asiaticoside into a cyclodextrin inclusion complex to significantly improve its solubility and bioavailability. However, the retention time of the single inclusion complex system in vivo is relatively short, resulting in limited sustained release of the drug. To solve this problem, the present invention further added the asiaticoside-cyclodextrin inclusion complex to a chitosan gel system to form a composite nanogel. This strategy can prolong the retention time of the drug at the skin site and improve the local absorption effect. However, when preparing the asiaticoside chitosan composite nanogel of the present invention, it was found that the amount of glutaraldehyde used had a great influence on the viscosity of the gel. As shown in Figure 5 A below, when the ratio of glutaraldehyde to chitosan was 80 μL:0.8 g, the gel had low viscosity, strong fluidity, and a short residence time on the skin; when the ratio of glutaraldehyde to chitosan was 100 μL:0.8 g, the viscosity was moderate and it was easy to coat ( Figure 5 as shown in B below); when the ratio of glutaraldehyde to chitosan was 120 μL:0.8 g, the viscosity was too high and it was difficult to coat evenly ( Figure 5 as shown in C below). Therefore, when preparing the asiaticoside chitosan composite nanogel of the present invention, it is necessary to control the amount of glutaraldehyde used.

[0105] In summary, the asiaticoside chitosan composite nanogel system shows significant potential in regulating the expression of acne-related inflammatory factors and reducing local skin inflammatory reactions. By improving the solubility and local bioavailability of asiaticoside, this system overcomes the limitations of its poor water solubility and short retention time, and is expected to provide a more effective treatment plan for acne and other skin inflammatory diseases. In the future, with the continuous optimization of technology, the application of this system in the field of local drug delivery will be further expanded, providing new ideas and innovative technical support for the treatment of skin diseases.

[0106] The embodiments described above are only descriptions of the preferred modes of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention should fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for preparing a product for treating acne, characterized in that: The following steps are involved: The asiatica glycoside solution is added to the β-cyclodextrin solution and stirred, and then the organic solvent is removed by rotary evaporation, followed by freeze drying to obtain an asiatica glycoside inclusion compound; After chitosan is dissolved in an acetic acid solution, polyvinyl alcohol is added to dissolve it, and then glutaraldehyde is added to carry out a cross-linking reaction to obtain a cross-linking solution; The asiatica glycoside inclusion compound, hydroxypropyl methylcellulose, ethylparaben and water are added to the cross-linked solution and stirred to obtain a product for treating acne.

2. The preparation method according to claim 1, characterized in that: The asiaticoside solution is prepared by dissolving asiaticoside in anhydrous ethanol; the β-cyclodextrin solution is prepared by dissolving β-cyclodextrin in water; the mass ratio of asiaticoside in the asiaticoside solution to β-cyclodextrin in the β-cyclodextrin solution is 1:(3-5).

3. The preparation method according to claim 1, characterized in that: When preparing the asiaticaside inclusion compound, the stirring temperature is room temperature and the stirring time is 15 to 17 hours.

4. The preparation method according to claim 1, characterized in that: The mass concentration of the acetic acid solution is 0.1%; the concentration of chitosan in the cross-linking solution is 8 mg / mL; the concentration of polyvinyl alcohol in the cross-linking solution is 30 mg / mL; the volume mass ratio of the glutaraldehyde to the chitosan is (80-120) μL:0.8 g, preferably 100 μL:0.8 g.

5. The preparation method according to claim 1, characterized in that: The mass ratio of asiaticaoside in the asiaticaoside solution to the chitosan is 5:

8.

6. The preparation method according to claim 1, characterized in that: The temperature for adding polyvinyl alcohol to dissolve is set to 80° C. After the polyvinyl alcohol is dissolved, the temperature drops to room temperature and then glutaraldehyde is added to carry out a cross-linking reaction; the cross-linking reaction is specifically carried out at room temperature for 25 to 35 minutes.

7. The preparation method according to claim 1, characterized in that: The concentration of chitosan in the product is 8 mg / mL.

8. The preparation method according to claim 1, characterized in that: The concentration of the hydroxypropyl methylcellulose in the product is 5 mg / mL; the concentration of the ethylparaben in the product is 0.5 mg / mL.

9. The product prepared according to the preparation method according to any one of claims 1 to 8.

10. Use of the product according to claim 9 in the preparation of a drug for treating acne.

Images