A kind of external-use lindera serrata gel for treating acne and its preparation method and application

Abstract

The present invention belongs to the field of application of traditional Chinese medicine, and specifically relates to a topical gel of Linderae medica for the treatment of acne, and a preparation method and application thereof. The present invention uses the ethanol extract of Linderae medica as the active part, and adopts modern preparation technology combined with transdermal penetration technology to prepare a topical gel, which can be used for the relief and treatment of acne infection through in vitro and in vivo pharmacodynamic evaluation and safety evaluation. Therefore, the present invention promotes the improvement of the traditional administration form of Linderae medica, broadens the clinical indications of Linderae medica, provides a new choice of medication for the clinical treatment of acne, and provides a reference for the repositioning research of Linderae medica combined with modern medicine, provides a scientific basis for the "new use of old medicine" of Linderae medica, and promotes the inheritance and innovation of traditional Chinese medicine with clinical application as the foothold.

Description

Technical Field

[0001] The invention belongs to the field of application of traditional Chinese medicines, and particularly relates to a Linderae umbellatae external-use gel for treating acne, and a preparation method and application thereof. Background Art

[0002] Acne is a chronic skin disease that occurs frequently in the pilosebaceous glands of the cheeks, chest, and back. The global prevalence rate is 9.4%, making it the eighth most prevalent disease and the third most common skin disease in the world. The pathogenesis of acne is complex, and is closely related to factors such as microbial infection, abnormal keratinization of the pilosebaceous gland ducts, endocrine levels, and excessive sebum secretion. Among them, colonization of Propionibacterium acnes is an important factor in causing acne. Currently, antibiotics, hormones, and retinoic acid drugs are the main treatments for acne in clinical practice. Although the symptoms of most patients can be partially relieved during treatment, there are still problems such as antibiotic resistance, high risk of recurrence, and large side effects, which limit their clinical application. With the integration of traditional Chinese medicine and modern science and technology, it has become an important research direction to explore safe and efficient functional components from natural plants to prevent and treat acne.

[0003] "Li Liao Pian Wen" says: "The principle of external treatment is the same as the principle of internal treatment, and the medicine for external treatment is the same as the medicine for internal treatment. The only difference is the method." External application of Chinese medicine is one of the characteristics of external treatment in traditional Chinese medicine. It has been continuously developed and summarized by medical scientists of all generations. It has unique advantages in treating skin diseases and is also an important way to treat acne.

[0004] Linderae radix is ​​the dried root of Linderae radix of the genus Linderae of the family Lauraceae. As a traditional and commonly used Chinese medicinal material, it has a long history of clinical application and has the effects of regulating qi and activating blood circulation, warming the middle and dispersing cold, regulating qi and relieving pain. In traditional medicine, Linderae radix is ​​mostly used in the form of decoction pieces or decoctions, with low bioavailability, poor stability, and inconvenience in taking, which limits the full play of its efficacy. In addition, the currently commercially available Linderae radix granules are clinically used for promoting qi and relieving pain, warming the kidney and dispersing cold, Suquan pills and Suquan capsules are mainly used for tonifying the kidney and reducing urine, and Linderae radix preparations for other indications have not been reported. With the development of modern preparation technology, the exploration of new indications for Linderae radix and the improvement of the form of administration have broad potential and necessity, which meet the development needs of the modernization and internationalization of traditional Chinese medicine.

[0005] Chinese medicine gel is a new type of external preparation with many advantages such as easy use, comfort, good biocompatibility, etc. It is suitable for skin and mucosal administration, which can not only avoid the influence of first-pass effect on oral administration, but also reduce the adverse reactions of drugs, which is in line with the concept of external treatment of internal diseases in traditional Chinese medicine. The preparation process of Chinese medicine gel is simple, and it can accommodate extremely fine powders and extracts of Chinese medicine compound, etc. It can effectively overcome the problem that Chinese medicine has a large molecular weight, poor extractability of effective components, and cannot penetrate the "skin brick wall structure" composed of stratum corneum and lipids to act on dermal cells and enter the blood circulation. It can be used as an option to improve traditional Chinese medicine external preparations. In the prior art, there is no preparation and evaluation method for external preparations of Linderae medica, and at present, there is no public report on the research of Linderae medica extracts and external preparations against acne infection at home and abroad. Summary of the invention

[0006] In view of the technical gaps in the prior art, the present invention provides a topical gel of Linderae medica for the treatment of acne. The present invention uses the ethanol extract of Linderae medica as the active part, and adopts modern preparation technology combined with transdermal penetration technology to prepare a topical gel, which can be used for the relief and treatment of acne infection after in vitro and in vivo pharmacodynamic evaluation and safety evaluation. Therefore, the present invention promotes the improvement of the traditional administration form of Linderae medica, broadens the clinical indications of Linderae medica, provides a new choice of medication for the clinical treatment of acne, and provides a reference for the repositioning research of Linderae medica combined with modern medicine, provides a scientific basis for the "new use of old medicine" of Linderae medica, and promotes the inheritance and innovation of traditional Chinese medicine with clinical application as the foothold.

[0007] The invention also provides a method for preparing the Linderae umbellatae external gel.

[0008] Another object of the present invention is to provide the use of the above-mentioned Linderae umbellatae external gel as a medicine for treating acne.

[0009] In order to achieve the above object, the technical solution adopted by the present invention is:

[0010] The invention provides a lindera serrata external gel for treating acne. Every 100 g of the gel comprises the following raw materials: 5 g to 20 g of lindera serrata ethanol extract, 1 g to 1.5 g of carbomer, 12 g to 18 g of glycerol, a pH regulator, 0.1 g of a preservative, and 0.5 g to 1.5 g of a penetration enhancer.

[0011] Preferably, the pH regulator is triethanolamine or sodium hydroxide; the amount of the pH regulator added is to adjust the pH value of the Linderae serrata external gel to 5.5-6.5; the preservative is ethyl hydroxybenzoate; and the penetration enhancer is menthol, borneol or laurocapram.

[0012] Most preferably, the penetration enhancer is menthol.

[0013] The present invention also provides a method for preparing the above-mentioned Linderae umbellatae external-use gel, comprising the following steps:

[0014] (1) Weigh an appropriate amount of carbomer and evenly disperse it in distilled water. After it is completely swollen, add glycerol and a pH adjuster while stirring to obtain a gel matrix;

[0015] (2) Take the ethanol extract of Linderae obesa, add ethanol solution and stir to completely dissolve it, which serves as the drug liquid phase; add the penetration enhancer and antibacterial agent to the ethanol solution and stir to completely dissolve them, which serves as the penetration enhancer liquid phase;

[0016] (3) Add the drug solution phase and the penetration-enhancing solution phase to the gel matrix in batches, add distilled water to make up the weight, add a pH adjuster, and stir until a brown-yellow transparent gel is formed.

[0017] Preferably, in step (1), the amount of distilled water added accounts for 50% of the prescription amount of the Linderae umbellatae external gel; preferably, in step (1) and step (3), the amount of pH regulator added is to adjust the pH to 5.5-6.5.

[0018] Preferably, in step (2), the concentration of the ethanol solution is 40%.

[0019] The invention also provides application of the Linderae umbellatae external gel as a medicine for treating acne.

[0020] Preferably, the Linderae serrata external gel is used as a drug for inhibiting Propionibacterium acnes.

[0021] In the external-use gel of Linderae rutaecarpa prepared by the present invention, the preparation process of the ethanol extract of Linderae rutaecarpa is as follows: 8 kg of Linderae rutaecarpa decoction pieces are taken, crushed and soaked in 8 times the amount of 80% ethanol overnight, and then heated and refluxed for extraction 4 times: the first extraction is for 2 hours, the second extraction is for 1.5 hours with 8 times the amount of 80% ethanol, and the third and fourth extractions are respectively for 1 hour with 5 times the amount of 80% ethanol, after the four extracts are combined, the ethanol is recovered under reduced pressure, and the concentrated solution is dried to obtain the ethanol extract;

[0022] Furthermore, the ethanol extract was dried with a freeze-drying yield of 5.8% and an active ingredient content of 6.5%; the water bath drying yield was 5.5% and an active ingredient content of 6.2%; the reduced pressure drying yield was 5.3% and an active ingredient content of 3.0%. The ethanol extracts of Linderae odorifera obtained by the three drying methods were used to prepare topical gels, and the cumulative transdermal penetration of the active ingredients was 404 μg, 388 μg, and 283 μg, respectively, and the intradermal retention was 239 μg, 225 μg, and 171 μg, respectively.

[0023] Furthermore, when the quality of the topical gel prepared by the present invention is evaluated, the percutaneous permeation of the active ingredient in the ethanol extract of the lindera medica meets the zero-order kinetic equation, and the mass concentration of the active ingredient plays a key role in the percutaneous permeation. As the permeation time increases, the permeation rate of the active ingredient first increases and then decreases. The percutaneous permeation of the active ingredient in the topical gel of the lindera medica meets the first-order kinetic equation, and the active ingredient permeates at a constant rate and is not affected by its own mass concentration, thereby achieving a sustained release effect.

[0024] Furthermore, the intradermal retention rates of the active ingredients in the topical gel and ethanol extract of Linderae obesa were 20.2% and 13.3%, respectively, indicating that making the ethanol extract of Linderae obesa into a gel can help improve the retention of the active ingredients in the skin, forming a drug reservoir and prolonging the efficacy.

[0025] Furthermore, when the external use gel of Linderae odorata prepared by the present invention was evaluated for its antibacterial effect in vitro, Propionibacterium acnes was used, and the concentration of the bacterial suspension was 1×10 6 CFU / mL, the minimum inhibitory concentration of the ethanol extract of Linderae umbellatae was 6.39 mg / mL, the minimum bactericidal concentration was 12.78 mg / mL, the minimum inhibitory concentration of the external gel of Linderae umbellatae was 25.57 mg / mL (approximately equivalent to 3.125 mg / mL of the ethanol extract of Linderae umbellatae), and the minimum bactericidal concentration was 50.13 mg / mL (approximately equivalent to 6.25 mg / mL of the ethanol extract of Linderae umbellatae).

[0026] Furthermore, 3.125 mg / mL of Linderae umbellata ethanol extract and 25.57 mg / mL of Linderae umbellata external gel were used to intervene in Propionibacterium acnes. Scanning electron microscopy observation showed that after the intervention of Linderae umbellata external gel, a large number of bacteria were severely wrinkled and twisted, severely damaged and broken, and adhesion substances were released. After the intervention of Linderae umbellata ethanol extract, the surface of the bacteria was rough and slightly wrinkled, and a small number of bacteria were twisted and deformed, indicating that the antibacterial effect of Linderae umbellata ethanol extract was enhanced when it was made into gel.

[0027] Furthermore, when the in vitro and in vivo pharmacodynamics evaluation of the topical gel of the present invention was performed, a mouse composite acne model induced by Propionibacterium acnes and oleic acid was established as an animal model to simulate the in vivo environment of acne. The modeling combined with treatment was used to evaluate the relief and treatment effects of the topical gel of the Wuyao on acne on the back of mice. 100 μL of 50% oleic acid was applied to the back of mice every day; Propionibacterium acnes (1×10 8CFU / mL) 50 μL, the dosage concentrations of Wuyao topical gel were 125 mg / g, 62.5 mg / g, and 31.25 mg / g, respectively, and 0.2 g / mouse was administered daily for 15 consecutive days. Compared with the model group, the high and medium dose groups of Wuyao topical gel can significantly improve the redness, swelling, papules and pustules in the acne area on the back of mice, significantly improve the pathological morphology of the acne area on the back, down-regulate the expression of inflammatory proteins in the back lesion tissue, and reduce the level of inflammatory factors in the serum of acne mice.

[0028] Furthermore, when the safety of the lindera serrata gel prepared by the present invention was evaluated, a skin irritation experiment was conducted using New Zealand rabbits, and a skin allergy experiment was conducted using guinea pigs to jointly evaluate the skin safety of the lindera serrata gel. The results showed that the lindera serrata gel was non-irritating and non-sensitizing.

[0029] Compared with the traditional drying method, the freeze-drying method of the ethanol extract of Linderae agglomerata provided by the present invention can effectively reduce the loss of heat-sensitive components, retain the effective ingredients in the medicine, and at the same time reduce the moisture content, reduce the growth of microorganisms, and improve the stability of the ethanol extract of Linderae agglomerata.

[0030] The preparation method of the lindera agglomerata external gel provided by the present invention can dissolve the lindera agglomerata ethanol extract with maximum efficiency without irritating the skin and destroying the active ingredients; through single factor test and response surface test, the gel matrix with uniform and transparent appearance, uniform texture, easy spread and moderate viscosity is obtained by continuous screening and optimization; the drug loading amount and the type and dosage of the penetration enhancer are screened by transdermal penetration technology, which can take into account the transdermal penetration and intradermal retention of the active ingredients at the same time, and is conducive to the local treatment of the skin external preparation.

[0031] The topical lindera atractylodes gel provided by the present invention changes the concentration-dependent permeation mechanism of the active ingredients in the lindera atractylodes atractylodes ethanol extract, can continuously and stably permeate the active ingredients into the lesion site, and at the same time improves the retention of the active ingredients in the skin, forming a drug reservoir to prolong the drug effect.

[0032] The external-use lindera serrata gel provided by the invention has a significantly enhanced inhibitory effect on Propionibacterium acnes compared with the lindera serrata ethanol extract.

[0033] The lindera serrata external gel provided by the present invention can effectively improve the pathological damage on the back of acne mice, reduce the content of inflammatory factors in the serum of acne mice, and down-regulate the expression of inflammatory proteins in acne lesions, compared with the third-generation retinoic acid inflammatory acne treatment drug adapalene gel, thereby providing an experimental basis and scientific basis for the application of lindera serrata external gel in the clinical treatment of acne.

[0034] The present invention provides an external Chinese medicine preparation for the treatment of acne and a preparation method thereof, and is used for the preparation of a topical gel of Linderae umbellatae and its pharmacodynamic evaluation against acne infection. It is firstly found that the ethanol extract of Linderae umbellatae has the effect of inhibiting Propionibacterium acnes; Linderae umbellatae is made into a skin topical preparation for the first time, and the in vitro antibacterial activity of the ethanol extract of Linderae umbellatae is enhanced, the transdermal mechanism of the ethanol extract of Linderae umbellatae is changed, and the transdermal penetration and intradermal retention of the active ingredients are improved; and it is firstly realized that the transdermal administration of Linderae umbellatae is used for the treatment of acne infection. It promotes the improvement of the traditional administration form of Linderae umbellatae, broadens the clinical indications of Linderae umbellatae, provides a new choice of medication for the clinical treatment of acne, provides a reference for the repositioning research of Linderae umbellatae combined with modern medicine to a certain extent, provides a scientific basis for the "new use of old medicine" of Linderae umbellatae, and promotes the inheritance and innovation of traditional Chinese medicine with clinical application as the foothold.

[0035] The beneficial effects of the present invention are:

[0036] 1. The present invention firstly discovered that the ethanol extract of Linderae umbellatae has the effect of inhibiting Propionibacterium acnes in vitro, enriched the pharmacological activity of Linderae umbellatae and provided an experimental basis for the development of Linderae umbellatae external preparations.

[0037] 2. The present invention is the first to prepare the ethanol extract of Linderae umbellatae into a topical gel, which significantly enhances the inhibitory effect on Propionibacterium acnes, changes the concentration-dependent penetration mechanism of the active ingredient, and can continuously and stably penetrate the active ingredient into the lesion site, while increasing the retention of the active ingredient in the skin, helping to form a drug reservoir and prolong the efficacy, providing a scientific basis for the effectiveness of Linderae umbellatae topical preparations.

[0038] 3. The topical gel of Linderae odoratum provided by the present invention can significantly improve the pathological morphology of acne on the back of mice induced by Propionibacterium acnes and oleic acid, reduce the content of inflammatory factors in the serum of acne mice, and down-regulate the expression of inflammatory proteins in acne lesions, providing a new medication option for the clinical treatment of acne.

[0039] 4. The present invention realizes the transdermal administration of Lindera medica for the first time, which is used for the improvement and treatment of skin diseases acne infection, promotes the improvement of the traditional administration form of Lindera medica, broadens the clinical indications of Lindera medica, provides a reference for the repositioning research of Lindera medica and the "new use of old medicine", and promotes the inheritance and innovation of traditional Chinese medicine with clinical application as the foothold.

[0040] 5. The present invention adopts freeze-drying to obtain the ethanol extract of Linderae agglomerata. Compared with traditional vacuum drying and water bath drying, the invention has high yield and high content of active ingredients. The active ingredients of the prepared external gel have better transdermal penetration and intradermal retention, which not only saves time cost but also improves the efficacy.

[0041] 6. The Linderae umbellatae external-use gel provided by the present invention has a simple preparation process, low cost, good spreadability, no greasy feeling, easy to clean, good transdermal absorption, good acne treatment effect, easy to promote and apply, and can be used as an option for improving the process of traditional Chinese medicine external-use preparations.

[0042] 7. The topical gel of Linderae odoratissimum provided by the present invention is mild to the skin and does not cause irritation or allergic reaction after repeated use. This is particularly important for acne patients who need long-term medication, especially when applied to sensitive parts such as the face, which helps to improve patient compliance. Its good tolerability also shows that in addition to short-term treatment, the product can be used in the maintenance stage after the disease is relieved, or even as part of daily skin care products. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 The response surface diagrams (A, B, C) and two-dimensional contour diagrams (a, b, c) of the interaction between glycerol, carbomer, and pH;

[0044] Figure 2 Permeation curves of active ingredients in topical gel containing different penetration enhancers;

[0045] Figure 3 Permeation curves of active ingredients in Linderae serrata topical gel with different dosages of menthol;

[0046] Figure 4 Comparison chart of antibacterial rate results;

[0047] Figure 5 Agar plate images: Linderae obesa external application gel group (A), adapalene gel group (B), Linderae obesa ethanol extract group (C) and blank control bacteria group (D);

[0048] Figure 6 SEM images of the morphology of Propionibacterium acnes: blank control bacteria (A), intervention with ethanol extract of Linderae odoratum (B), intervention with adapalene gel (C) and intervention with topical Linderae odoratum gel (D);

[0049] Figure 7 Morphological changes of the back skin of mice in each group;

[0050] Figure 8 Pathological changes of the back skin tissue of mice in each group; (A) blank control group; (B) model group; (C) adapalene gel positive control group; (D) low-dose group of Wuyao topical gel; (E) medium-dose group of Wuyao topical gel; (F) high-dose group of Wuyao topical gel;

[0051] Fig. 9 Changes in serum inflammatory factor levels of mice in each group;

[0052] Fig.10The expression level of TNF-α protein in the back skin tissue of mice in each group; (A): blank control group; (B): model group; (C): adapalene gel positive control group; (D): low-dose group of Wuyao topical gel; (E): medium-dose group of Wuyao topical gel; (F): high-dose group of Wuyao topical gel;

[0053] Fig.11 The expression level of IL-1β protein in the back skin tissue of mice in each group; (A): blank control group; (B): model group; (C): adapalene gel positive control group; (D): low-dose group of Wuyao topical gel; (E): medium-dose group of Wuyao topical gel; (F): high-dose group of Wuyao topical gel

[0054] Fig.12 The expression level of IL-6 protein in the back skin tissue of mice in each group; (A): blank control group; (B): model group; (C): adapalene gel positive control group; (D): low-dose group of Wuyao topical gel; (E): medium-dose group of Wuyao topical gel; (F): high-dose group of Wuyao topical gel

[0055] Fig.13 Skin irritation test: Inspection of the skin appearance at the administration site;

[0056] Fig.14 HE staining of the skin at the administration site in the skin irritation test. DETAILED DESCRIPTION

[0057] The present invention is further described below in conjunction with specific examples, which are provided to better illustrate the content of the present invention rather than to limit the present invention. The experimental methods used in the examples are conventional methods unless otherwise specified; the materials and reagents used can be obtained from commercial sources unless otherwise specified. Therefore, non-essential improvements and adjustments made by those skilled in the art based on the above-mentioned invention content still fall within the scope of protection of the present invention.

[0058] Example 1 Single factor test to screen prescriptions

[0059] 1. Preparation process and scoring criteria of Linderae serrata topical gel

[0060] Weigh an appropriate amount of carbomer and evenly disperse it in 50% of the prescription amount of distilled water, stir and swell naturally overnight. After complete swelling, stir and add an appropriate amount of glycerin, adjust the pH value, and make it a transparent gel matrix. Take an appropriate amount of ethanol extract of Linderae rutaecarpa, add 40% ethanol and stir to completely dissolve it, as the drug liquid phase. Take an appropriate amount of penetration enhancer and antibacterial agent, add 40% ethanol and stir to completely dissolve it, as the penetration-enhancing liquid phase. Stir the drug liquid phase and the penetration-enhancing liquid phase into the blank matrix in batches, add distilled water to 100 g, adjust the pH value, and stir for a long time in the same direction until a crystal clear, delicate and uniform brown-yellow transparent gel is formed.

[0061] Table 1 Gel scoring criteria

[0062]

[0063] The specific scoring method is:

[0064] Comprehensive score = appearance score + spreadability score + stability score + viscosity score

[0065] Stability score = (heat resistance score + cold resistance score + cold resistance score + centrifugal score) / 4

[0066] 2. Carbomer dosage screening

[0067] Carbomer is the main component of the gel matrix and has the functions of thickening, suspending, stabilizing the system, regulating the release of water and active substances, etc. The carbomer dosage was selected as 0.8%, 1.0%, 1.25%, 1.5%, and 2.0%, respectively, and other preparation conditions remained unchanged. The effect of carbomer dosage on the gel was evaluated according to the gel scoring criteria shown in Table 1.

[0068] As shown in Table 2, when the amount of carbomer is 0.8%, the gel has greater fluidity and poorer stability. When the amount of carbomer is 2.0%, the gel is too viscous and difficult to apply, and the dissolution is insufficient, with a granular feel and reduced gloss. Properly increasing the amount of carbomer can help improve the viscosity and stability of the gel matrix. Therefore, the most suitable amount of carbomer is 1.0% to 1.5%.

[0069] Table 2 Carbomer dosage screening results

[0070]

[0071] 3. Glycerol dosage screening

[0072] Glycerol, as a moisturizer in the formulation, can increase the contact area between carbomer and water, and promote and stabilize the swelling of carbomer. The glycerol dosage was selected as 10%, 12%, 15%, 18%, and 20%, respectively, and other preparation conditions remained unchanged. The effect of glycerol dosage on the gel was evaluated according to the gel scoring standard.

[0073] As shown in Table 3, the difference in the amount of glycerol is mainly reflected in the viscosity. As the amount of glycerol increases, the viscosity of the gel increases first and then decreases. Therefore, the most suitable amount of glycerol added is between 12% and 18%.

[0074] Table 3 Screening results of glycerol dosage

[0075]

[0076] 4. pH screening

[0077] Carbomer contains a large number of carboxyl groups in its structure, which will significantly reduce the pH value of the gel and cause damage to the skin. In this study, triethanolamine was used to adjust the pH value. The pH values ​​were adjusted to 5.0, 5.5, 6.0, 6.5, and 7.0 respectively. Other preparation conditions remained unchanged, and the effect of pH value on the gel was evaluated according to the gel scoring standard.

[0078] As shown in Table 4, pH value mainly affects the spreadability and viscosity of the gel. As the pH value increases, the viscosity of the gel increases and the fluidity decreases. When the pH value reaches 5.0, the gel begins to become viscous; when the pH value is 6.5, the spreadability and viscosity decrease slightly but do not affect the use; and when the pH value is 7.0, the gel is too viscous and difficult to spread. Therefore, the most suitable pH range is 5.5-6.5.

[0079] Table 4 pH screening results

[0080]

[0081] Example 2 Response surface experiment to optimize prescription

[0082] 1. Response Surface Experimental Design

[0083] Based on the results of the single-factor experiment, three factors, namely, glycerol dosage (A), carbomer dosage (B) and pH value (C), were selected as independent variables, and the comprehensive score of the gel was used as the response value. The Design-Expert 13 software was used to perform a three-factor three-level response surface experimental design to further optimize the matrix formulation. The specific experimental factors and levels are shown in Table 5.

[0084] Table 5 Factor levels and coding of response surface experiment

[0085]

[0086] 2. Results and Discussion

[0087] The gel matrix formulation was subjected to Box-Behnken design using Design-Expert 13 software (see Table 6). 17 groups of gel matrices were prepared according to the experimental design, and the best gel matrix formulation was selected based on the comprehensive score of the gel matrix.

[0088] Table 6 Box-Behnken experimental design results

[0089]

[0090] Furthermore, the Design Expert 13 software was used to fit the data obtained in Table 6 with a quadratic polynomial regression model to predict the response value, and the quadratic polynomial expression was obtained:

[0091] R =9.44-0.025 A +0.2125 B -0.3125 C -0.05 AC +0.025 BC -0.5575 A 2 -0.6325 B 2 -1.48 C 2 .

[0092] R For comprehensive rating; A is the dosage of Carbomer; B is the amount of glycerol used; C is the pH value.

[0093] Furthermore, the quadratic multinomial regression model was subjected to variance analysis, and the results are shown in Table 7. Confidence of the model P <0.01, the difference is extremely significant, indicating that the model is meaningful; the correlation coefficient R 2 =0.9794, correction factor R 2 Adj =0.9954, indicating that the model fits well, the predicted value has a high correlation with the measured value, and can well describe the experimental results and has good predictive power. P The value is 0.2643 (>0.05), and the difference is not significant, indicating that the probability of the model prediction value not fitting the actual value is very low. The F value of the model reflects the influence of the factor on the comprehensive score of the gel. The larger the F value, the greater the influence. B , C and the quadratic term A 2 , B2 , C 2 Corresponding P The values ​​were all less than 0.0001, indicating that there were significant interactions among the factors.

[0094] Table 7 Response surface variance analysis table

[0095]

[0096] The response surface 3D map and the two-dimensional contour map can intuitively reflect the values ​​of each factor under the optimal conditions and the strength of the interaction between the factors. The more elliptical and dense the two-dimensional contour map is, the stronger the interaction between the factors is; the larger the response value is, the steeper the response surface 3D map is, and the more obvious the interaction between the factors is. Figure 1 It can be seen that the order of influence of each factor on the comprehensive score of gel matrix is: C > B > A , that is, pH value > carbomer > glycerol. At the same time, the optimal gel matrix prescription was predicted to be 14.95% glycerol, 1.29% carbomer, pH value 5.95, and the comprehensive score was 9.474 points.

[0097] Example 3 Drug loading screening

[0098] Based on the optimal gel matrix prescription of Example 2 above, topical gels of Linderae umbellatae with drug loadings of 5%, 10%, 12.5%, 15%, and 20% were prepared respectively, and other preparation conditions remained unchanged. The effect of drug loading on the gel was evaluated according to the gel scoring standard.

[0099] As the drug loading increases, the amount of solvent, carbomer and triethanolamine also needs to be increased. The remaining water in the prescription is not enough to fully swell the carbomer, resulting in a large number of micelles mixed into the matrix, the gel has a granular appearance, and the stability is reduced. As shown in Table 8, when the drug loading is 10% and 12.5%, the gel has a uniform and delicate appearance, is easy to apply, has good stability, and has the highest comprehensive score. In order to take into account the content of the active ingredient, the most suitable drug loading is 12.5%.

[0100] Table 8 Drug loading screening results

[0101]

[0102] Example 4 Screening of penetration enhancer types

[0103] Based on the optimal gel matrix prescription and the most suitable drug loading, menthol, borneol and laurocapram were selected as penetration enhancers to prepare Linderae serrata gel for external use. The in vitro percutaneous permeation technology was used, and the sesquiterpenoid active ingredients with high content and good permeability in Linderae serrata gel were used as evaluation indicators. Combined with the comprehensive scoring criteria of the gel, the type and dosage of the penetration enhancers were screened.

[0104] 1. Preparation of topical gel containing different types of penetration enhancers

[0105] Menthol, borneol and laurocapram were selected as penetration enhancers respectively, and other preparation conditions remained unchanged to prepare 0% and 1% penetration enhancers of Linderae obesa external gel. The ethanol extract solution of Linderae obesa was used as a reference to investigate the effects of different penetration enhancers on the percutaneous permeation of the active ingredients in Linderae obesa external gel, and the cumulative permeation per unit area of ​​the active ingredients was calculated ( Q n ) 、 Cumulative permeability ( Q ) 、 Transdermal rate ( J ) 、 Permeation enhancement factor ( ER ) and intradermal retention rate, draw the in vitro permeation curve, and fit the permeation curve with an equation.

[0106] 2. In vitro percutaneous permeation test method

[0107] The in vitro percutaneous permeation experiment was conducted using a transdermal diffusion instrument. The water bath temperature was (32 ± 0.2) °C, the magnetic rotor speed was (300 ± 1.0) rpm, the receiving cell volume was 12 mL, and the effective diffusion area was 1.766 cm 2 . Use tweezers to fix the skin of male Bama Xiang pigs with a quantitative loop between the receiving pool and the supply pool, with the skin stratum corneum facing the supply pool. There is about 0.35 g of Linderae obesa external gel in the quantitative loop (equivalent to 43 mg of Linderae obesa ethanol extract), and 1 mL of Linderae obesa ethanol extract solution (43 mg / mL) in the supply pool. Add transdermal receiving solution to the scale in the receiving pool and remove all bubbles to make the receiving solution close to the skin. Take 1.0 ml of transdermal receiving solution samples at 2, 4, 8, 12, 18, and 24 hours, and add an equal volume of receiving solution at the same temperature.

[0108] 3. Determination of transdermal receiving solution and intradermal active ingredient content

[0109] Chromatographic conditions: Chromatographic column: ACE EXCEL C18-PFP (2.1×100 mm, 3 μm); Mobile phase A: 0.1% formic acid in water; Mobile phase B: 0.1% formic acid in methanol; Flow rate: 0.5 mL / min; Column temperature: 40°C; Injection volume: 2.0 μL. See Table 9 for chromatographic elution conditions.

[0110] Table 9 Gradient elution table

[0111]

[0112] The mass spectrometry conditions used a heated electric spray ionization (HESI) source, a sheath gas flow rate of 60 arb, an auxiliary gas flow rate of 20 arb, a spray voltage of 3.5 kV, an ion transfer tube temperature of 350 °C, and an auxiliary gas heater temperature of 400 °C. The mass spectrometry scanning mode was positive ion parallel reaction monitoring (PRM), and the resolution was 35000 FWHM. The mass spectrometry fragmentation information and parameters of the active substances are shown in Table 10.

[0113] Table 10 Mass spectrometry fragment information and parameters of each compound

[0114]

[0115] Solution preparation

[0116] Reference substance solution: Accurately weigh 5 mg of each active ingredient reference substance, dissolve it in a blank solvent (50% methanol) and quantitatively dilute it to make a solution containing approximately 250 ng of each substance per 1 mL as the reference substance stock solution; accurately measure an appropriate amount of each substance and dilute it with a solvent to make a linear series of reference substance solutions of different concentrations.

[0117] Test solution of Linderae umbellatae external gel: take about 0.25 g of Linderae umbellatae external gel, accurately weigh it, put it in a 50 mL volumetric flask, add appropriate amount of solvent, ultrasonicate at 40℃ for 2 hours to dissolve and dilute to the scale, shake well, filter, accurately measure 5 mL of the filtrate, put it in a 50 mL volumetric flask, dilute to the scale with solvent, shake well, and obtain.

[0118] Transdermal penetration test sample solution: Take the transdermal receiving solution of the transdermal penetration experiment of Linderae umbellatae external gel and filter it to obtain the solution.

[0119] Intradermal retention test solution: Take the skin after the percutaneous penetration experiment of Linderae umbellatae external gel, wash it with physiological saline to remove all residual drugs, cut it into pieces and put it in a centrifuge tube, add 5 mL of 40% ethanol to fully homogenize, make up to 10 mL with methanol, centrifuge at 12000 rpm for 10 min, filter the supernatant, and take the filtrate to obtain the solution.

[0120] 4. In vitro percutaneous permeation test evaluation parameters

[0121] (1) Cumulative infiltration per unit area ( Q n )

[0122] Q n =

[0123] in, C n is the mass concentration of the active ingredient in the receiving solution measured at the nth sampling point (μg / mL), V is the volume of the receiving pool (12 mL), C i is the mass concentration of the active ingredient in the receiving solution measured at the n-1th sampling point (μg / mL), V i is the sampling volume (1 mL), A is the effective penetration area (1.766 cm 2 ), C i V i It is the cumulative amount of drug lost due to sampling.

[0124] (2) Cumulative permeability ( Q )

[0125] Q = Q n × A / W ×100%

[0126] in, W It is the content of active ingredients in the Linderae odorifera external gel.

[0127] (3) Transdermal rate J )

[0128] Cumulative permeability per unit area Q n Penetration time t Perform linear regression, and the slope of the regression equation is the transdermal rate.

[0129] (4) Penetration enhancement factor ( ER )

[0130] ER = J e / J 0

[0131] in, J e is the rate of drug penetration after using a penetration enhancer; J 0 It is the transdermal rate of the drug without penetration enhancer.

[0132] 5. Results and Discussion

[0133] As shown in Table 11, compared with the topical gel without penetration enhancer, borneol, menthol and laurocapram all promote the penetration of the active ingredients in the topical gel to varying degrees. Figure 2 It can be seen that the percutaneous penetration of the active ingredients in the topical gel of Linderae serrata conforms to the zero-order kinetic equation. r 2 The value is closest to 1, indicating that the active ingredients in the topical gel of Linderae obesa penetrate into the receiving solution at a constant rate and are not affected by the mass concentration of the active ingredients themselves, thus achieving a sustained-release effect; while the percutaneous permeation of the active ingredients in the ethanol extract of Linderae obesa is consistent with the first-order kinetic equation, indicating that the mass concentration of the active ingredients plays a key role in their percutaneous permeation. Q n The trend is that the concentration of active ingredients in the ethanol extract of Linderae odoratissimum is increased rapidly at first and then slowly. Therefore, making the ethanol extract of Linderae odoratissimum into a gel changes the concentration-dependent penetration mechanism of the active ingredients, and can continuously and stably penetrate the active ingredients into the lesion site.

[0134] As shown in Table 13, compared with other penetration enhancers, the active ingredient of Linderae umbellatae external gel containing menthol is Q 24 The highest value was 473.59 μg / cm 2 , ER The highest is 2.78, Q The highest is 45.11%, J The highest is 21.644 μg / cm 2 / h, and the highest intradermal retention rate was 20.22%. Compared with the ethanol extract solution of Linderae umbellatae, the active ingredient of Linderae umbellatae external gel containing menthol Q 24 Although low, the intradermal retention rate is high, indicating that the preparation of the ethanol extract of Linderae agglomerata into a gel can help improve the retention of active ingredients in the skin, forming a drug reservoir to prolong the efficacy. In summary, the topical Linderae agglomerata gel containing menthol has both good skin permeability and good drug retention ability, so menthol was finally selected as a penetration enhancer.

[0135] Table 11 The active ingredients in the topical gel containing different penetration enhancers Q n ( n = 3)

[0136]

[0137] Table 12 In vitro permeation curve equation fitting of active ingredients in topical gel containing different permeation enhancers

[0138]

[0139] Table 13 Transdermal permeation test parameters of active ingredients in topical gel containing different penetration enhancers

[0140]

[0141] Example 5 Screening of the dosage of penetration enhancer

[0142] 1. Preparation of topical gel with different dosages of penetration enhancer

[0143] Taking menthol as a penetration enhancer and keeping other preparation conditions unchanged, topical gels containing 0.5%, 0.7%, 1%, 1.3%, and 1.5% menthol were prepared. The effect of menthol dosage on the percutaneous permeation of active ingredients was investigated by in vitro percutaneous permeation experiments. Q n , Q, J, ER The in vitro permeation curve was plotted and the equation was fitted for the permeation curve. The appearance, spreadability, viscosity and stability of the gel were also investigated.

[0144] 2. Results and Discussion

[0145] From Table 14 and Figure 3 It can be seen that the increase in the amount of menthol Q n When the dosage of menthol increases to 1.3%, the active ingredient Q n It starts to decrease, indicating that menthol only promotes the penetration of active ingredients within a certain dosage range, and excessive dosage inhibits penetration. As shown in Table 15, the penetration of active ingredients in the topical gel of Linderae odoratissimum with different dosages of menthol all conforms to the zero-order kinetic equation, that is, constant rate penetration. Q 24 The highest value was 473.59 μg / cm 2 , Q The highest is 45.11%, J The fastest is 21.644 μg / cm 2 / h, ER The highest is 2.78, and the intradermal retention rate is 20.22%, which is second only to the topical gel containing 1.3% menthol. In addition, it can be seen from Table 16 that the topical gel containing 1% menthol has good appearance, spreadability, viscosity and stability. Therefore, the most suitable dosage of menthol is determined to be 1%.

[0146] Table 14 The active ingredients in the topical gel of Linderae odorifera with different dosages of menthol Q n ( n = 3)

[0147]

[0148] Table 15 In vitro transdermal test parameters of active ingredients in different dosages of menthol Linderae serrata topical gel

[0149]

[0150] Table 16 Menthol dosage screening results

[0151]

[0152] Example 6 Preparation method of Linderae serrata external gel

[0153] Take 1.29g of carbomer, accurately weigh it, and evenly disperse it in 50g of distilled water. After stirring, swell naturally overnight. After complete swelling, stir and add 14.95g of glycerol, and adjust the pH value to 5.95 with triethanolamine to make it a transparent gel matrix. Take another 12.5g of Linderae ethanol extract, accurately weigh it, add 16.5g of solvent [40% ethanol] and stir it to completely dissolve it as the drug liquid phase. Take another 1g of menthol and 0.1g of ethylparaben, add 2.5g of solvent [40% ethanol] and stir it to completely dissolve it as the penetration-enhancing liquid phase. Stir the drug liquid phase and the penetration-enhancing liquid phase into the gel matrix in batches, add distilled water to 100g, adjust the pH value to 5.95 with triethanolamine, and stir for a long time in the same direction until a crystal clear, delicate and uniform brown-yellow transparent gel is formed.

[0154] Based on the Linderae umbellatae external application gel prepared in Example 6, subsequent efficacy tests were performed:

[0155] Example 7 Comparative study on the in vitro antibacterial activity of Linderae serrata external gel and marketed acne treatment drugs 1

[0156] 1. Determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of drugs

[0157] Take a 96-well plate and set up a growth control group, a blank control group, a negative control group and a concentration gradient group. The growth control group is a mixture of 200 μL of culture medium and 20 μL of Propionibacterium acnes suspension, the blank control group is 200 μL of culture medium, the negative control group is 200 μL of different concentrations of test solution, and the concentration gradient group is a mixture of 200 μL of different concentrations of test solution and 20 μL of Propionibacterium acnes suspension. Each group is set up with 3 replicate wells. Place in an anaerobic box and culture at 37℃ in a constant temperature shaking incubator for 24-48 h. Detect the absorbance at 600 nm and calculate the inhibition rate. The inhibition rate formula is: (growth control well-blank control well)-(drug well-negative control well) / (growth control well-blank control well)×100%. According to the inhibition rate results, combined with the naked eye observation of the solution and precipitation in the well, the lowest concentration of the drug solution for inhibition, that is, the minimum inhibitory concentration, is comprehensively judged.

[0158] Take 100 μL of the culture solution containing bacteria and drugs that is higher than the minimum inhibitory concentration and spread it on a solid plate, place it in an anaerobic box and culture it in a constant temperature shaking incubator at 37°C for 24-48 hours. Observe whether there is bacterial growth, and determine the mass concentration of the plate count ≤5 colonies as the minimum bactericidal concentration of the drug.

[0159] 2. Results and Discussion

[0160] After the culture was completed, the culture medium of the blank control group in the 96-well plate was clear, indicating that there was no contamination by foreign bacteria and the culture medium was stable. The culture medium of the growth control group was turbid, indicating that the strain grew well and had no effect on the evaluation of antibacterial effect. The ethanol extract of Linderae odoratissimus, Linderae odoratissimus topical gel and adapalene gel, a marketed drug for the treatment of acne, all showed significant inhibitory effects on Propionibacterium acnes in vitro.

[0161] As shown in Table 17, the minimum inhibitory concentration of the topical gel of Linderae serrata is lower than that of the ethanol extract of Linderae serrata, which is 25.57 mg / mL (approximately equivalent to 3.125 mg / mL of the ethanol extract of Linderae serrata), and the minimum inhibitory concentration of adapalene gel, a marketed drug for the treatment of acne, is 25.40 mg / mL. This indicates that the inhibitory effect of the topical gel of Linderae serrata on Propionibacterium acnes is better than that of the ethanol extract of Linderae serrata, which is basically consistent with that of adapalene gel.

[0162] Table 17 Comparison of MIC and MBC results

[0163]

[0164] like Figure 4As shown in the figure, the antibacterial rates of 25 mg / mL (equivalent to 3.125 mg / mL of ethanol extract of Linderae obesa) and 3.125 mg / mL of ethanol extract of Linderae obesa were 82.7% and 59.0%, respectively, and the antibacterial rate of 25 mg / mL of adapalene gel was 85.2%. The results of plate coating at this concentration are shown in the figure. Figure 5 shown. Figure 5 In the Wuyao topical gel group ( Figure 5 The number of colonies on the plate of A) was significantly higher than that on the ethanol extract of Linderae obesum ( Figure 5 C) group, compared with the adapalene gel group ( Figure 5 There is no significant difference in B).

[0165] Example 8 Comparative study on the in vitro antibacterial activity of Linderae serrata external gel and marketed acne treatment drugs 2

[0166] 1. Bacterial morphological observation

[0167] About 15.63 mg of Linderae umbellata ethanol extract, about 0.125 g of Linderae umbellata external gel (equivalent to 15.63 mg of Linderae umbellata ethanol extract), and about 0.125 g of adapalene gel were taken respectively, diluted to 5 mL with a suspension of Propionibacterium acnes in the logarithmic growth phase, and placed in an anaerobic box and cultured in a constant temperature shaking incubator at 37°C for 48 h. The culture medium was centrifuged at 7000 rpm for 5 min to collect the bacteria; the cells were washed, dispersed and centrifuged with PBS solution, and the precipitate was fixed with 2.5% glutaraldehyde solution at room temperature for 2-3 h and fixed at 4°C overnight; the cells were rinsed with PBS solution for 5 times, each time for 15 min, and fixed with 1% osmium acid in the dark for 1.5 h; the cells were rinsed with PBS solution twice and ultrapure water three times, each time for 15 min; the cells were dehydrated with gradient concentrations of ethanol (50%, 70%, 80%, 90%, 100%, 100%) for 15 min each time, and gold was sprayed after sufficient drying. The morphological changes of bacteria after different drug interventions were observed under a scanning electron microscope.

[0168] 2. Results and Discussion

[0169] like Figure 6 As shown, untreated bacteria ( Figure 6 A) in the figure has a smooth surface, is complete and full, and has a short rod-shaped body, slightly curved, with blunt ends. The body treated with the ethanol extract of Linderae odoratum ( Figure 6 B) has a rough and slightly wrinkled surface, and a small number of bacteria are twisted and deformed; while the bacteria ( Figure 6 C and Figure 6 (D) A large number of bacteria were severely wrinkled and twisted, damaged and broken, and had adhesive substances released.

[0170] The above antibacterial activity research results show that the inhibitory effect of the topical gel of Linderae serrata on Propionibacterium acnes is significantly stronger than that of the ethanol extract of Linderae serrata. The research team's previous rheological studies on the topical gel of Linderae serrata showed that it has good mechanical stability and viscoelasticity, and in vitro percutaneous permeation studies have shown that it has a sustained release and long-acting property. Therefore, it is speculated that the topical gel of Linderae serrata is easier to combine with bacteria, thereby increasing the contact area, and by slowly releasing the drug, increasing the drug concentration on the bacterial surface, thus showing a stronger antibacterial effect than the ethanol extract of Linderae serrata.

[0171] Example 9 Evaluation of the therapeutic effect of Linderae serrata external application gel on complex acne on the back of mice

[0172] 1. Animal grouping, modeling and drug administration

[0173] After 1 week of adaptive feeding, 60 male ICR mice were depilated on their backs and randomly divided into a blank group, a model group, an adapalene gel control group (1 mg / g), a high-dose group (125 mg / g), a medium-dose group (62.5 mg / g), and a low-dose group (31.25 mg / g) of Wuyao topical gel, with 10 mice in each group.

[0174] Every morning, except for the blank group, 100 μL of 50% oleic acid (diluted with liquid paraffin) was applied to the back of mice in other groups, and Propionibacterium acnes (1×10 8 CFU / mL) 50 μL; every night, except for the model group, the blank group was given 0.2 g of gel matrix, the adapalene gel control group was given 0.2 g of adapalene gel, and the high, medium, and low dose groups of Wuyao topical gel were given 0.2 g of Wuyao topical gel of corresponding specifications, which were evenly applied on the back of mice for 15 consecutive days. Starting from the first day of modeling and drug administration, the morphological changes of the back skin of mice were observed every five days.

[0175] 2. Specimen Collection

[0176] 12 hours after the last administration, blood was collected from the eyeballs and centrifuged at 3000 rpm and 4°C for 15 min to prepare serum, which was stored at -80°C for subsequent detection of inflammatory factors. The model skin on the back of the mice was completely peeled off and fixed in 4% paraformaldehyde for pathological section analysis and immunohistochemical examination.

[0177] 3. Effect of Wuyao topical gel on the back skin morphology of mice with complex acne model

[0178] Skin morphology changes after 15-day combined treatment of acne on the back of mice Figure 7As shown. Blank group: The skin tissue on the back of the mice was soft, the surface was delicate and smooth, and it was light pink. The subcutaneous capillaries were clear and not dilated. Model group: Compared with the blank group, the hair was sparse, curly and falling off. The skin on the head, face, ears and back showed wrinkles, thickening and swelling. A large number of sebaceous glands fell off. The epidermis was rough and covered with a large amount of keratin and scabs. The subcutaneous capillaries were obviously dilated. There were obvious pustules, papules, acne, ulcers, scabs and other skin lesions. The base of the lesions was dark red, and there was no obvious exudate. Each treatment group: On the 5th day of medication, the skin of mice in each treatment group showed varying degrees of wrinkles and roughness, shedding of sebaceous glands, keratinization and scabs, and capillary dilation; on the 10th day of medication, the epidermis of mice in the adapalene gel positive control group was covered with a thick layer of keratinized crust, the hair was sparse, curly and falling, and the skin under the hair was dark red. The epidermis of mice in the high-dose group of Wuyao topical gel was covered with a thin layer of keratinization, and the skin was rough and light pink after the scab fell off, without obvious papules and pustules. The epidermis of mice in the medium and low-dose groups of Wuyao topical gel was covered with a thin layer of keratinization, the skin under the keratinization and hair was dark red, and there was capillary dilation under the skin, with small papules and pustules On the 15th day of medication, the keratinized skin and scabs on the head, face, ears and back of the mice in the high-dose group of Wuyao topical gel and the positive control group of adapalene gel were basically shed, the hair on the back skin was basically lost, the new skin was soft, delicate and smooth, and was light pink. There was no dilation of capillaries under the skin, and there were scattered mild papules, pustules and erythema. A small amount of keratinized skin and scabs were visible on the back of the mice in the medium-dose group of Wuyao topical gel, and the new skin was slightly red and swollen and very thin. Dilation of capillaries could still be seen under the skin, and small papules, pustules and nodules still existed. A large amount of keratinized skin and scabs were still visible on the back of the mice in the low-dose group of Wuyao topical gel, and the skin was slightly red and swollen, the subcutaneous capillaries were obviously dilated, and there were still papules, pustules and erythema.

[0179] 4. Pathological section examination

[0180] The back skin tissue of mice was sectioned and stained using hematoxylin-eosin (HE) staining method, and the pathological state of the skin was observed under a microscope. Figure 8As shown. Blank control group: No obvious abnormalities were found in the back skin of mice, the stratum corneum was thin, the hair follicles were of normal volume and less content, the hair follicle wall did not proliferate, the sebaceous glands were of normal volume, the boundaries between the dermis, epidermis and subcutaneous tissue were clear, the collagen fibers in the dermis were spindle-shaped and arranged tightly and orderly, and no pathological changes such as inflammatory cell infiltration and capillary dilation were found in the subcutaneous tissue. Model group: Compared with the blank control group, the skin tissue structure was destroyed, the epidermal granular layer and spinous layer cells proliferated, the dermis fibrous tissue cells degenerated and necrotized with inflammatory cell infiltration, capillary dilation, and congestion. Drug-administered groups: Compared with the model group, the skin tissue structure of the adapalene gel positive control group was basically restored, with clear boundaries between the epidermis, dermis and subcutaneous tissue, and the collagen fibers in the dermis were spindle-shaped and arranged tightly and orderly. Occasionally, slight inflammatory cell infiltration was observed, and new capillaries were formed; in the low-dose group of the Wuyao topical gel, the boundaries between the epidermis, dermis and subcutaneous tissue became slightly clearer, the epidermis was hyperkeratotic, and the fibroblasts in the dermis were degenerated and necrotic with inflammatory cell infiltration; in the medium-dose group of the Wuyao topical gel, the skin tissue structure was basically restored to normal, the epidermis was hyperkeratotic, cell fragments were visible in the epidermis, and inflammatory cell infiltration was occasionally observed; in the high-dose group of the Wuyao topical gel, the subcutaneous tissue layers were clearly demarcated, the epidermis and dermis were thinned, cell fragments were visible in the epidermis, the subcutaneous collagen fibers were arranged more regularly, and inflammatory cell infiltration was occasionally observed. Compared with the low- and medium-dose groups of the test substance, the lesions were mild and narrow in scope.

[0181] 5. Determination of inflammatory factors in mouse serum

[0182] The levels of TNF-α, IL-6 and IL-1β in mouse serum were detected by enzyme-linked immunosorbent assay. Fig. 9 As shown in the figure, compared with the blank group, the levels of TNF-α, IL-1β and IL-6 in the serum of the model group mice were significantly increased. Compared with the model group, the levels of TNF-α, IL-1β and IL-6 in each drug-treated group decreased to varying degrees, indicating that the Wuyao topical gel can reduce the level of inflammatory cytokines in the serum of mice, among which the high-dose Wuyao topical gel group had a similar inhibitory effect to the adapalene gel positive control group.

[0183] 6. Detection of inflammatory protein expression in mouse back skin lesions

[0184] Immunohistochemistry was used to detect the expression of TNF-α, IL-1β and IL-6 proteins in the back skin tissue of mice. Figure 10-12As shown in the figure, the area and color of the brown-yellow particles indicate positive expression. Compared with the blank group, the expression of TNF-α, IL-1β and IL-6 in the back skin tissue of mice in the model group was significantly increased; compared with the model group, the expression of TNF-α, IL-1β and IL-6 in the back skin tissue of mice in each drug-treated group was reduced to varying degrees, indicating that Wuyao gel can inhibit the expression of inflammatory proteins. The positive control group of adapalene gel and the high-dose group of Wuyao topical gel were particularly significant.

[0185] Example 10 Skin irritation test of Linderae umbellatae external gel

[0186] 1. Animal grouping and treatment

[0187] Eight New Zealand rabbits, half male and half female, that had been adapted to feeding for one week were randomly divided into an intact skin group and a damaged skin group, with 4 rabbits in each group. The hair on both sides of the back spine was trimmed 24 hours before the experiment, and there was no epidermal damage. The hair removal area was about 3 cm × 3 cm, and the application area was about 2.5 cm × 2.5 cm. After hair removal, the damaged skin group was scratched with a No. 12 needle. The degree of skin damage was limited to the damage to the epidermis, and the degree of scratching was based on bleeding.

[0188] 2. Animal Dosing

[0189] Using the same body left and right self-comparison method, 0.5 g of Linderae medica topical gel (125 mg / g) was applied to the right skin, and 0.5 g of gel matrix was applied to the left skin. Wrap it with two layers of gauze and one layer of plastic wrap, and then fix it with a non-irritating medical non-woven patch. The application time was 6 hours. Apply once a day for 7 consecutive days. After each application, clean the administration site with warm water to remove residual drugs. After the last administration, wash with warm water to remove residual drugs, and observe the skin of the administration site for erythema, edema, etc. at 1, 24, 48, and 72 hours. After the end, the skin of the administration site was taken for pathological section analysis.

[0190] 3. Evaluation Criteria

[0191] The skin irritation of the topical gel of Wuyao was evaluated by the irritation intensity of the administration site and pathological tissue sections. The skin irritation reaction scoring standard is shown in Table 18, and the skin irritation intensity evaluation standard is shown in Table 19.

[0192] Table 18 Skin irritation reaction scoring criteria

[0193]

[0194] Table 19 Evaluation criteria for skin irritation intensity

[0195]

[0196] 4. Results and Discussion

[0197] The results of changes in the skin appearance of New Zealand rabbits at the administration site before and after administration are shown in Fig.13 HE staining results are shown in Fig.14 The results of the skin irritation reaction score are shown in Table 20. From the appearance pictures, it can be seen that before and after administration, there was no obvious change in the skin of the intact skin group and the damaged skin group at the administration site, no erythema and edema appeared, and there was no significant difference from the skin on the blank matrix side; from the HE staining results, the stratum corneum of the intact skin group was intact, and no obvious histopathological changes such as inflammatory cell infiltration and tissue edema were found in the dermis; the damaged skin in the damaged skin group showed thickening of the epidermis, focal inflammatory cell infiltration in the dermis, and connective tissue hyperplasia, and no obvious histopathological changes were found in the non-damaged skin, and there was no significant difference from the skin on the blank matrix side. This shows that repeated administration of Wuyao topical gel has no irritating reaction to the skin.

[0198] Table 20 Skin irritation intensity score

[0199]

[0200] Example 11 Skin allergy test of Linderae umbellatae external gel

[0201] 1. Animal grouping and treatment

[0202] Thirty healthy guinea pigs were randomly divided into a blank control group, a 2,4-dinitrochlorobenzene group, and a Linderae scabra topical gel group, with 10 in each group, half male and half female. 24 h before the experiment, both sides of the back of the guinea pigs were depilated, the depilated area was about 3.0 cm × 3.0 cm, and the application area was about 2.5 cm × 2.5 cm.

[0203] 2. Animal Dosing

[0204] Sensitization contact administration: 0.2 mL of 1.0% 2,4-dinitrochlorophenylpropiophenone solution was applied to the skin of the left hair removal area of ​​the guinea pigs in the positive control group, 0.2 g of blank matrix was applied to the blank control group, and 0.2 g of lindera serrata gel (125 mg / g) was applied to the lindera serrata gel group, covered with two layers of gauze and one layer of plastic wrap, and medical non-woven fabric was wrapped around the guinea pig's body for 2 circles to fix it. After 6 hours, warm water was used to remove the residual drug and wipe the skin clean. The same method was repeated once on the 7th day and the 14th day respectively.

[0205] Provocative contact administration: On the 14th day after the last sensitization, 0.2 mL of 0.1% 2,4-dinitrochlorophenylpropiophenone solution was applied to the right hair removal area of ​​the guinea pigs in the positive control group, 0.2 g of blank matrix was applied to the blank control group, and 0.2 g of lindera serrata gel (125 mg / g) was applied to the lindera serrata gel group, covered with two layers of gauze and one layer of plastic wrap, and medical non-woven fabric was wrapped around the guinea pig's body for 2 circles to fix it. After 6 hours, the residual drug was removed with warm water and the skin was wiped clean. Observe immediately and observe the skin sensitization again at 24, 48, and 72 hours.

[0206] 3. Evaluation Criteria

[0207] Skin sensitization was scored. The scoring criteria for skin allergic reactions are shown in Table 21. The evaluation criteria for skin allergy are shown in Table 22. The incidence of allergic reactions was calculated according to the following formula.

[0208] Allergic reaction rate (%) = × 100%

[0209] Table 21 Scoring criteria for skin allergic reactions

[0210]

[0211] Table 22 Evaluation criteria for skin sensitization

[0212]

[0213] 4. Results and Discussion

[0214] In the sensitization control group, 0.1% 2,4-dinitrochlorobenzene was used as an irritant. Guinea pig skin showed different degrees of erythema and edema allergic reactions at 24 h, 48 h, and 72 h after stimulation, and the incidence of allergic reactions was 70%, 60%, and 30%, respectively. According to the skin sensitization evaluation standard, it was judged to be highly sensitized, indicating that the sensitization control group was established; no systemic allergic reactions such as asthma and standing instability were observed. In the guinea pigs in the Wuyao topical gel group and the blank control group, no erythema and edema allergic reactions occurred at the administration site 24 h, 48 h, and 72 h after stimulation, and the sensitization rate was 0%. No systemic allergic reactions such as asthma and standing instability were observed, indicating that Wuyao topical gel did not produce sensitization reactions on guinea pig skin. The results are shown in Table 23.

[0215] Table 23 Skin allergic reaction results

[0216]

Claims

1. A topical gel for the treatment of acne, characterized in that: Including the following ingredients: 5g-20g of Linderae umbellatae ethanol extract, 1g-1.5g of carbomer, 12g-18g of glycerol, pH regulator, 0.1g of preservative, 0.5g-1.5g of penetration enhancer; The preparation method of the ethanol extract of Linderae rutaecarpa is as follows: 8 kg of Linderae rutaecarpa decoction pieces are taken, crushed, soaked in 80% ethanol in 8 times the amount overnight, and then heated and refluxed for extraction 4 times: the first extraction is for 2 hours, the second extraction is for 1.5 hours with 8 times the amount of 80% ethanol, and the third and fourth extractions are respectively for 1 hour with 5 times the amount of 80% ethanol, after the four extracts are combined, the ethanol is recovered under reduced pressure, and the concentrated solution is freeze-dried to obtain the ethanol extract; The pH regulator is triethanolamine or sodium hydroxide; the amount of the pH regulator added is to adjust the pH value of the lindera serrata external gel to 5.5-6.5; the preservative is ethyl hydroxybenzoate; and the penetration enhancer is menthol.

2. A method for preparing the Linderae umbellatae external gel according to claim 1, characterized in that: The following steps are involved: (1) Weigh an appropriate amount of carbomer and evenly disperse it in distilled water. After it is completely swollen, add glycerol and a pH adjuster while stirring to obtain a gel matrix; (2) Take the ethanol extract of Linderae obesa, add ethanol solution and stir to completely dissolve it, which serves as the drug liquid phase; add the penetration enhancer and ethylparaben and stir to completely dissolve them, which serves as the penetration enhancer liquid phase; (3) Add the drug solution phase and the penetration-enhancing solution phase to the gel matrix in batches, add distilled water to make up the weight, add a pH adjuster to adjust the pH value, and stir until a brown-yellow transparent gel is formed.

3. The preparation method according to claim 2, characterized in that: In step (1), the amount of distilled water added accounts for 50% of the prescription amount of the Linderae umbellatae external gel.

4. The preparation method according to claim 2, characterized in that: In step (2), the concentration of the ethanol solution is 40%.

5. The preparation method according to claim 2, characterized in that: In step (1) and step (3), the amount of pH adjuster added is to adjust the pH to 5.5-6.

5.

6. Use of the Linderae umbellatae external application gel as claimed in claim 1 in the preparation of a medicine for treating acne.

7. The use according to claim 6, characterized in that: The application of the Linderae serrata external gel in preparing a medicine for inhibiting Propionibacterium acnes.

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