Nano-micelle of cryptotanshinone wrapped by glycyrrhizic acid as well as preparation method and application of nano-micelle

Abstract

The invention provides a glycyrrhizic acid coated cryptotanshinone nano-micelle as well as a preparation method and application thereof, and relates to the technical field of skin care products. According to the glycyrrhizic acid-coated cryptotanshinone nano-micelle, glycyrrhizic acid is used as a carrier for carrying cryptotanshinone, and glycyrrhizic acid and cryptotanshinone cooperate with each other to jointly play antibacterial, anti-inflammatory and anti-lipid secretion roles, so that the glycyrrhizic acid-coated cryptotanshinone nano-micelle has an obvious synergistic effect; meanwhile, through preparation of the nano-micelle, the problems that existing cryptotanshinone is poor in water solubility and stability and difficult to apply to a water system cosmetic formula are effectively relieved. In addition, detection verifies that compared with single use of cryptotanshinone, the glycyrrhizic acid coated cryptotanshinone nano-micelle can effectively reduce the toxicity of cryptotanshinone to keratinocytes, can intervene in various main pathogenesis of acne, and has antibacterial, acne-removing and repairing effects.

Description

Technical Field

[0001] The invention relates to the technical field of skin care products, and in particular to a nano-micelle of cryptotanshinone encapsulated with glycyrrhizic acid, and a preparation method and application thereof. Background Art

[0002] Acne is a chronic inflammatory skin disease of the hair follicle sebaceous gland unit, commonly known as acne and blackheads. It is one of the most common skin problems and often occurs in areas rich in sebaceous glands such as the face, neck, chest and back.

[0003] Related studies have shown that the formation of acne is related to excessive secretion of sebaceous glands, excessive bacterial growth, clogged pores, endocrine factors, genetics, diet and lifestyle habits, environmental factors, improper use of cosmetics, etc. At the same time, due to the increase in androgens that promote sebum production, the imbalance between sebum production and secretion leads to sebum blockage in the hair follicles. Further excessive lipid secretion and excessive keratinization in the hair follicle funnel cause sebum accumulation, leading to an increase in Propionibacterium acnes around the hair follicles, which decomposes triglycerides in sebum, produces free fatty acids, and at the same time chemotactics inflammatory cells and mediators, ultimately inducing and aggravating inflammatory responses.

[0004] So far, many compositions and methods have been proposed for improving acne problems in acne-removing cosmetics. For example, Chinese patent application CN202311440027.8 discloses a tanshinone derivative and its preparation method and application. Cryptotanshinone is added to mPEG activated ester, and a new hydrophilic tanshinone derivative is prepared through stirring, dialysis and other steps, which can solve the problems of excessive sebum secretion, keratinization of hair follicles, pore blockage, acne-related bacterial infection, etc. Patent application CN117357429A relates to an anti-acne composition and a method of use. The cosmetic composition generally includes a cosmetically acceptable carrier and a skin active ingredient combination including sulfur, niacinamide, and phenethylresorcinol, which can be used to treat acne, treat inflammation, and provide anti-aging benefits to the skin. However, the above-disclosed method for preparing tanshinone derivatives is too cumbersome, involving continuous stirring for 12 hours and dialysis for 48 hours. At the same time, the above method uses cosmetic banned ingredients such as dichloromethane for preparation, and there may be safety issues such as organic solvent residues.

[0005] Cryptotanshinone, derived from the traditional Chinese medicinal material Danshen, has broad application prospects in the cosmetics field due to its potential anti-inflammatory, antioxidant and acne-removing effects. However, in actual application, cryptotanshinone faces the dual challenges of poor water solubility and insufficient stability. These technical defects directly affect its performance and application effect in cosmetics.

[0006] First, as a fat-soluble ingredient, cryptotanshinone has extremely low solubility in water, which makes it difficult to evenly disperse and dissolve cryptotanshinone when preparing water-based cosmetics, thus affecting the stability and use effect of the product. For example, when preparing anti-acne essences or creams, the poor water solubility of cryptotanshinone will cause stratification, precipitation or crystallization in the product, which not only affects the appearance and texture of the product, but may also reduce its acne-removing effect. In patent CN109045109A, the inventors tried to improve the dispersibility of cryptotanshinone in water by adding surfactants. However, although this method can improve the dispersibility of cryptotanshinone to a certain extent, the use of surfactants may also have a potential impact on the mildness and safety of the product.

[0007] Second, cryptotanshinone is susceptible to photochemical hydrolysis and oxidative degradation under the influence of environmental factors such as light, temperature and pH value, resulting in reduced activity or even failure. This instability not only limits the long-term preservation of cryptotanshinone in cosmetics, but may also lose its acne-removing effect due to environmental changes during use. Especially in water-based cosmetics, due to the presence of water, the stability problem of cryptotanshinone is more prominent. In patent CN115192585A, the inventors attempted to improve the stability of cryptotanshinone by adding antioxidants and sunscreens. However, although this method can extend the shelf life of cryptotanshinone to a certain extent, the addition of antioxidants and sunscreens may also increase the cost and complexity of the product.

[0008] In addition, the anti-acne cosmetics or anti-acne products currently on the market have limited therapeutic effects and are prone to recurrence or side effects such as dry skin and desquamation, and still cannot fully meet consumers' needs to improve acne problems.

[0009] In contrast, the glycyrrhizic acid encapsulation technology proposed in the present application can not only effectively improve the water solubility of cryptotanshinone, but also maintain the mildness and safety of the product; at the same time, the glycyrrhizic acid encapsulation technology proposed in the present application can effectively protect cryptotanshinone from environmental factors such as light, temperature and pH value by forming a stable complex, thereby improving its stability.

[0010] In view of this, the present invention is proposed. Summary of the invention

[0011] The purpose of the present invention is to provide a glycyrrhizic acid-encapsulated cryptotanshinone nanomicelle and a preparation method thereof. The nanomicelle uses glycyrrhizic acid as a carrier, and adopts a thin film dispersion hydration method to encapsulate cryptotanshinone to obtain the nanomicelle. Based on the mechanism of acne, the synergy of the glycyrrhizic acid carrier and cryptotanshinone is used to improve the efficacy of cryptotanshinone in treating acne from multiple angles, effectively solving the problems of poor water solubility and stability of cryptotanshinone, and difficulty in applying it to a water-soluble cosmetic formula system, thereby further enriching the market for anti-acne cosmetic raw materials.

[0012] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted: The present invention provides a glycyrrhizic acid-encapsulated cryptotanshinone nano-micelle, wherein the nano-micelle is mainly formed by self-assembly of glycyrrhizic acid and cryptotanshinone in water; The glycyrrhizic acid interacts with cryptotanshinone to form nano micelles in which cryptotanshinone is encapsulated in the glycyrrhizic acid using the glycyrrhizic acid as a carrier.

[0013] Furthermore, the raw materials for preparing the nano micelles include: cryptotanshinone, glycyrrhizic acid and water, and the ratio of cryptotanshinone, glycyrrhizic acid and water is 1g:10~20g:800~1200ml.

[0014] Furthermore, the ratio of cryptotanshinone, glycyrrhizic acid and water is 1g:15-20g:800-1200ml; Furthermore, the water is purified water.

[0015] Furthermore, the particle size of the nanomicelles is 4-15 nm, and the polydispersity index PDI is 0.235-0.392.

[0016] The present invention provides a method for preparing nano-micelles of cryptotanshinone encapsulated with glycyrrhizic acid, the preparation method comprising: Cryptotanshinone and glycyrrhizic acid are sequentially added to a solvent to be dissolved, and then the solvent is evaporated and recovered to obtain a film; the film is hydrated and dispersed using a film hydration method to obtain water-soluble glycyrrhizic acid-encapsulated cryptotanshinone nano-micelles.

[0017] Furthermore, the solvent includes at least one of anhydrous ethanol, dichloromethane and chloroform.

[0018] Furthermore, the method of evaporating and recovering the solvent is a rotary evaporation method; The parameters of the rotary evaporation method include: a rotary evaporation temperature of 50-60° C., a rotation speed of 80-120 rpm / min, a vacuum degree of 40-100 kPa, and a time of 20-30 min.

[0019] Further, the thin film hydration method comprises: Purified water is added into the film, followed by hydration, dissolution, incubation and ultrasonic dispersion in sequence to obtain water-soluble glycyrrhizic acid-encapsulated cryptotanshinone nano-micelles.

[0020] Furthermore, the hydration and dissolution incubation temperature is 45-80°C and the time is 30-90 min; Preferably, the temperature of the hydration dissolution incubation is 50-60°C and the time is 50-70 minutes; Furthermore, the frequency of the ultrasonic dispersion is 20-80 kHz, and the time is 10-30 min; Preferably, the frequency of the ultrasonic dispersion is 40-60 kHz, and the time is 10-15 min.

[0021] The invention provides an application of the glycyrrhizic acid-encapsulated cryptotanshinone nano-micelles in the preparation of acne prevention and treatment products.

[0022] Compared with the prior art, the present invention has the following beneficial effects: The glycyrrhizic acid-encapsulated cryptotanshinone nano-micelles provided by the present invention use glycyrrhizic acid as a carrier for carrying cryptotanshinone, and cooperate with cryptotanshinone to simultaneously exert antibacterial, anti-inflammatory, and anti-lipid secretion effects, and have obvious synergistic effects, effectively alleviating the problem that the existing cryptotanshinone has poor water solubility and poor stability, and is difficult to be applied to cosmetic formulations of water systems. Compared with the use of cryptotanshinone alone, the glycyrrhizic acid-encapsulated cryptotanshinone nano-micelles of the present invention have been tested and verified to effectively reduce the toxicity of cryptotanshinone to keratinocytes, and can intervene in a variety of major pathogenesis mechanisms of acne, and have antibacterial, acne-removing and repairing effects.

[0023] The invention provides a method for preparing glycyrrhizic acid-encapsulated cryptotanshinone nano-micelles, wherein glycyrrhizic acid is used as a carrier, and a thin film dispersion hydration method is used to encapsulate cryptotanshinone inside the glycyrrhizic acid to form water-soluble glycyrrhizic acid-encapsulated cryptotanshinone nano-micelles. The preparation method has the technical advantages of simple processing technology and easy operation.

[0024] The glycyrrhizic acid-encapsulated cryptotanshinone nano-micelles provided by the present invention can be widely used in the preparation process of acne prevention and treatment products. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0026] Figure 1 This is a particle size distribution diagram of the glycyrrhizic acid-encapsulated cryptotanshinone nanomicelles prepared in Example 1 of the present invention; Figure 2 This is an infrared spectrum (FT-IR) of the glycyrrhizic acid-encapsulated cryptotanshinone nanomicelles prepared in Example 1 of the present invention; Figure 3 This is an infrared spectrum (FT-IR) of the product prepared in Comparative Example 1 of the present invention; Figure 4 The experimental results of the Propionibacterium acnes inhibition zone of Example 1 and Comparative Example 2 provided in Experimental Example 4 of the present invention are shown in FIG. Figure 5 It is a local observation picture of the treatment of acne in each test group of the acne mouse model provided in Experimental Example 5 of the present invention; Figure 6 This is a graph showing changes in acne swelling volume in each experimental group of the acne mouse model provided in Experimental Example 5 of the present invention after acne treatment. DETAILED DESCRIPTION

[0027] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0028] According to one aspect of the present invention, a nano-micelle of cryptotanshinone encapsulated with glycyrrhizic acid, wherein the nano-micelle is mainly formed by self-assembly of glycyrrhizic acid and cryptotanshinone in water; The glycyrrhizic acid interacts with cryptotanshinone to form nano micelles in which cryptotanshinone is encapsulated in the glycyrrhizic acid using the glycyrrhizic acid as a carrier.

[0029] The glycyrrhizic acid-encapsulated cryptotanshinone nanomicelles provided by the present invention use glycyrrhizic acid as a carrier for carrying cryptotanshinone, act synergistically with cryptotanshinone, and simultaneously exert antibacterial, anti-inflammatory, and anti-lipid secretion effects, thereby effectively alleviating the problems of existing cryptotanshinone, such as poor water solubility and stability, and difficulty in application in cosmetic formulations of water systems.

[0030] It should also be noted that the carrier glycyrrhizic acid in this application is an amphiphilic active ingredient derived from the root of the traditional Chinese medicine licorice, containing two molecules of glucuronic acid and one molecule of glycyrrhetinic acid, which is water-soluble and can self-assemble with fat-soluble components to form nano-micelles, which are also called nano-vesicles. In addition, studies have confirmed that glycyrrhizic acid is one of the most important active ingredients in licorice, with antiviral, anti-inflammatory, antioxidant, immunomodulatory, and liver-protecting effects.

[0031] Therefore, the present application uses glycyrrhizic acid as a carrier to effectively alleviate the problems of poor water solubility and poor stability of existing cryptotanshinone, while also being able to exert its own anti-inflammatory effect, thereby exerting the characteristics and advantages of "drug-adjuvant integration". In addition, compared with the use of cryptotanshinone alone, the antibacterial, anti-inflammatory, and anti-lipid secretion effects of the nano-micelles of the present invention all have better effects. It has been tested and verified that the toxicity of cryptotanshinone to keratinocytes can be reduced, and glycyrrhizic acid can act as a carrier while synergizing with cryptotanshinone, and can effectively intervene in a variety of major pathogenesis of acne, with antibacterial, acne-removing and repairing effects.

[0032] In a preferred embodiment of the present invention, the raw materials for preparing the nano micelles include: cryptotanshinone, glycyrrhizic acid and water, and the ratio of cryptotanshinone, glycyrrhizic acid and water is 1g:10-20g:800-1200ml.

[0033] In the above preferred embodiment, the ratio of cryptotanshinone, glycyrrhizic acid and water is 1g:15~20g:800~1200ml; In a preferred embodiment of the present invention, the water is purified water.

[0034] In a preferred embodiment of the present invention, the particle size of the nanomicelles is 4-15 nm, and the polydispersity index PDI is 0.235-0.392.

[0035] According to one aspect of the present invention, a method for preparing nano-micelles of cryptotanshinone encapsulated with glycyrrhizic acid comprises: Cryptotanshinone and glycyrrhizic acid are sequentially added to a solvent to be dissolved, and then the solvent is evaporated and recovered to obtain a film; the film is hydrated and dispersed using a film hydration method to obtain water-soluble glycyrrhizic acid-encapsulated cryptotanshinone nano-micelles.

[0036] The invention provides a method for preparing glycyrrhizic acid-encapsulated cryptotanshinone nano-micelles, wherein glycyrrhizic acid is used as a carrier, and a thin film dispersion hydration method is used to encapsulate cryptotanshinone inside the glycyrrhizic acid to form water-soluble glycyrrhizic acid-encapsulated cryptotanshinone nano-micelles. The preparation method has the technical advantages of simple processing technology and easy operation.

[0037] In a preferred embodiment of the present invention, the solvent includes at least one of anhydrous ethanol, dichloromethane and chloroform.

[0038] In a preferred embodiment of the present invention, the method of evaporating and recovering the solvent is a rotary evaporation method; The parameters of the rotary evaporation method include: a rotary evaporation temperature of 50-60° C., a rotation speed of 80-120 rpm / min, a vacuum degree of 40-100 kPa, and a time of 20-30 min.

[0039] In a preferred embodiment of the present invention, the thin film hydration method comprises: Purified water is added into the film, followed by hydration, dissolution, incubation and ultrasonic dispersion in sequence to obtain water-soluble glycyrrhizic acid-encapsulated cryptotanshinone nano-micelles.

[0040] In the above preferred embodiment, the temperature of the hydration dissolution incubation is 45-80°C and the time is 30-90 min; Preferably, the temperature of the hydration dissolution incubation is 50-60°C and the time is 50-70 minutes; In the above preferred embodiment, the frequency of the ultrasonic dispersion is 20 to 80 kHz, and the time is 10 to 30 minutes; Preferably, the frequency of the ultrasonic dispersion is 40-60 kHz, and the time is 10-15 min.

[0041] Preferably, the glycyrrhizic acid-encapsulated cryptotanshinone nanomicelles and the preparation method thereof include: S1: dissolving cryptotanshinone in a solvent, adding glycyrrhizic acid, and using ultrasound to assist dissolution if necessary, to obtain an orange-red, clear and transparent mixture solution; S2: The mixture solution is subjected to rotary evaporation to recover the solvent, so as to disperse the mixture solution into a uniform film; S3: Add purified water to the dispersed film system, perform hydration, dissolution and incubation, and perform ultrasonic dispersion to obtain water-soluble glycyrrhizic acid-encapsulated cryptotanshinone nano-micelles.

[0042] According to one aspect of the present invention, the above-mentioned glycyrrhizic acid-encapsulated cryptotanshinone nanomicelles are used in the preparation of acne prevention and treatment products.

[0043] The glycyrrhizic acid-encapsulated cryptotanshinone nano-micelles provided by the present invention can be widely used in the preparation process of acne prevention and treatment products.

[0044] In a preferred embodiment of the present invention, the acne prevention and treatment product is a drug having at least one or more of antibacterial, acne-removing or repairing effects; And / or, the anti-acne product is a cosmetic having at least one or more of antibacterial, acne-removing or repairing effects.

[0045] The technical solution of the present invention will be further described below in conjunction with embodiments.

[0046] Example 1 A nano-micelle of cryptotanshinone coated with glycyrrhizic acid and a preparation method thereof, wherein glycyrrhizic acid is used as a carrier and cryptotanshinone is coated by a thin film dispersion hydration method, specifically comprising the following steps: S1: Accurately weigh 10 mg of cryptotanshinone and 150 mg of glycyrrhizic acid, and fully dissolve them in 15 mL of anhydrous ethanol solution to obtain a mixed solution of cryptotanshinone and glycyrrhizic acid, which is a clear orange-red liquid.

[0047] S2: The mixture was rotary evaporated at 70 kPa, 55° C. and 100 rpm for 30 min to remove ethanol and form a uniform film on the wall of the eggplant-shaped bottle of the rotary evaporator.

[0048] S3: Add 10 mL of purified water in small amounts and multiple times at 55°C to completely dissolve the film in water, incubate at 55°C for 60 min, disperse ultrasonically at 50 kHz for 15 min, filter, and finally obtain an orange-red clear solution.

[0049] In this embodiment, the ratio of cryptotanshinone, glycyrrhizic acid and water is 10 mg:150 mg:10 ml, and the converted ratio is 1 g:15 g:1000 ml.

[0050] Embodiment 2~5 Except for the difference in the ratio of cryptotanshinone, glycyrrhizic acid and water, Examples 2 to 5 are the same as Example 1. The ratio of cryptotanshinone, glycyrrhizic acid and water in Examples 2 to 5 is shown in Table 1.

[0051] Table 1:

[0052] Embodiment 6-10 Except that the parameters of the rotary evaporation in step S2 are different from those in Example 1, the other parameters of Examples 6 to 10 are the same as those in Example 1. The parameters of the rotary evaporation in Examples 6 to 10 are shown in Table 2.

[0053] Table 2:

[0054] Embodiment 11 The difference between this embodiment and embodiment 1 is that the hydration and dissolution incubation time in step S3 is 30 minutes, and the other parts are the same as embodiment 1.

[0055] Example 12 The difference between this embodiment and embodiment 1 is that the hydration and dissolution incubation time in step S3 is 90 minutes, and the other parts are the same as embodiment 1.

[0056] Comparative Examples 1 to 5 Comparative Examples 1 to 5 are the same as Example 1 except that the composition and ratio of cryptotanshinone, glycyrrhizic acid and water are different from those of Example 1. See Table 3 for cryptotanshinone, glycyrrhizic acid and water in Comparative Examples 1 to 5.

[0057] Table 3:

[0058] Comparative Example 6 The difference between this comparative example and Example 1 is that the rotation speed of the rotary evaporation in step S2 is 30 rpm, and the other parts are the same as Example 1.

[0059] Comparative Example 7 The difference between this comparative example and Example 1 is that the time of rotary evaporation in step S2 is 10 min, and the other parts are the same as Example 1.

[0060] Comparative Example 8 The difference between this comparative example and Example 1 is that the hydration and dissolution incubation time in step S3 is 10 min, and the other parts are the same as Example 1.

[0061] Comparative Example 9 The difference between this comparative example and Example 1 is that the hydration and dissolution incubation temperature in step S3 is 40° C., and the other parts are the same as Example 1.

[0062] Comparative Example 10 The difference between this comparative example and Example 1 is that the ultrasonic dispersion time in step S3 is 5 min, and the other parts are the same as Example 1.

[0063] The following experiment is carried out on the glycyrrhizic acid-encapsulated cryptotanshinone nanomicelles prepared by the present invention to further illustrate the advantages of the present invention.

[0064] Experimental Example 1 Particle size and potential experiment of cryptotanshinone nanomicelles encapsulated with glycyrrhizic acid Experimental method: The particle size and polydispersity coefficient of glycyrrhizic acid-encapsulated cryptotanshinone nanomicelles were determined by Malvern laser particle size analyzer. The instrument parameters during the measurement process were: test temperature 25°C; measurement position 4.65 nm; attenuator set to 8. Zeta potential measurement directly selected undiluted nanomicelle samples for measurement, data output, and the results were analyzed, as shown in Tables 4 and 5 below.

[0065] Table 4:

[0066] Table 5:

[0067] Based on the above, it can be seen that the particle size and Zeta potential of the glycyrrhizic acid-encapsulated cryptotanshinone nanomicelles prepared in Examples 1 to 12 of the present application are relatively small, with a particle size of 4 to 15 nm and a polydispersity index PDI of 0.235 to 0.392.

[0068] Figure 1This is the particle size distribution diagram of the glycyrrhizic acid-encapsulated cryptotanshinone nanomicelles prepared in Example 1 of the present invention.

[0069] However, since Comparative Example 1 does not contain cryptotanshinone, i.e. lacks the main active ingredient, it cannot exert good antibacterial and anti-inflammatory effects in the subsequent experimental evaluations; Since Comparative Example 2 does not contain glycyrrhizic acid, i.e. lacks the coating carrier glycyrrhizic acid, and cryptotanshinone cannot be dissolved in water, a suspension is obtained according to the process; In Comparative Example 3, due to the low content of glycyrrhizic acid in the coating carrier, cryptotanshinone could not be completely coated in the carrier glycyrrhizic acid, and the particle size result was too large (209.57±24.68 nm); In Comparative Example 4, due to the excessive glycyrrhizic acid content of the coated carrier, the excessive amount of coated carriers will self-assemble into blank glycyrrhizic acid micelles, resulting in a high PDI value (0.609±0.138); In Comparative Example 5, due to the excessively high content of purified water added, glycyrrhizic acid could not reach the critical micelle concentration and could not encapsulate cryptotanshinone, and the excessively high solvent ratio increased the application cost; In comparative examples 6 and 7, due to the low rotation speed or time of rotary evaporation, the particle size distribution during the dissolution of the drug film in step S3 was uneven; In Comparative Example 8, the drug film cannot be completely dissolved in step S3 due to the short hydration and dissolution incubation time; In Comparative Example 9, the temperature of the hydration dissolution incubation was too low, resulting in the inability to completely dissolve the drug film in step S3; In Comparative Example 10, the particle size distribution was uneven and relatively large (0.609±0.138) due to the short ultrasonic dispersion time.

[0070] Experimental Example 2 Characterization of glycyrrhizic acid-encapsulated cryptotanshinone nanomicelles by Fourier transform infrared spectroscopy (FT-IR) Experimental method: Fourier transform infrared spectroscopy (FT-IR) was used to measure the infrared absorption spectrum of glycyrrhizic acid-encapsulated cryptotanshinone nanomicelles.

[0071] Specifically, the samples prepared in Example 1, Example 2, Example 3, Comparative Example 1, Comparative Example 2, and Comparative Example 3 were mixed with 200 mg KBr, ground evenly, placed in an infrared tabletting mold and pressed into thin sheets. Different samples were collected by scanning 32 times, with a wavelength of 4000-400 cm-1 and a resolution of 2. The experimental results can be seen in Figure 2 and Figure 3 .

[0072] Figure 2 This is an infrared spectrum (FT-IR) of the glycyrrhizic acid-encapsulated cryptotanshinone nanomicelles prepared in Example 1 of the present invention.

[0073] Figure 3 This is an infrared spectrum (FT-IR) diagram of the product prepared in Comparative Example 1 of the present invention.

[0074] Comparing the results of Example 1 with those of Comparative Example 1, it was found that the infrared absorption characteristic peak of the glycyrrhizic acid-encapsulated cryptotanshinone nano-micelles at 3442.61 cm-1 was red-shifted to 3423.20 cm-1. -1 The change of FTIR characteristic absorption peaks proves that cryptotanshinone is successfully encapsulated by glycyrrhizic acid.

[0075] Experimental Example 3 The synergistic anti-Propionibacterium acnes effect of cryptotanshinone encapsulated by glycyrrhizic acid was demonstrated by the MIC of Propionibacterium acnes.

[0076] Experimental methods: (1) Configuration of samples to be tested: Samples were prepared using the experimental methods of Example 1, Comparative Example 1, and Comparative Example 2, respectively.

[0077] (2) Activation and preparation of strains: Propionibacterium acnes (ATCC 11827) strain was purchased from Guangdong Microbiological Culture Collection Center (GDMCC). 11.4 g RCM medium and 3.6 g agar powder were weighed into a clean conical flask, and 300 ml of distilled water was added to heat and dissolve. After complete dissolution, the strain was sterilized at 121°C for 30 min and set aside. The Propionibacterium acnes strain was inoculated into the RCM medium and cultured at 37°C for 20-28 hours for activation. The experiment was started after the third generation.

[0078] (3) MIC experiment: The Propionibacterium acnes preserved bacteria were inoculated into the reinforced clostridial medium (RCM), cultured at 37°C, activated, and a small amount of bacterial moss was picked and diluted to 0.5 McFarland units with sterile saline to prepare an inoculum suspension, which was sealed and refrigerated for later use. Each row tested a drug concentration, and a 2 mL EP tube was used to test the MIC value of the extract of the plant compound composition for inhibiting Propionibacterium acnes in the preparation example and the comparative preparation example.

[0079] The experiment was carried out using the microdilution method. 500 μL of culture medium was added to all 2 mL EP tubes, and then 500 μL of the plant compound inhibiting Propionibacterium acnes combination liquid was added to the first well, 500 μL of the mixed solution was drawn from the first well and added to the second well, and so on to the twelfth well, to obtain 2, 4, 8, 16, 32, 64, 128, 256, 512, 1024, 2048, and 4096 times diluted drugs, and 500 μL of the liquid was discarded in the twelfth well, and then 500 μL of the inoculated bacterial suspension was added to each of the 1-13 wells, among which, well 13 was the growth group and well 14 was the blank group. The growth of Propionibacterium acnes was observed after culturing at a constant temperature of 37°C for 24 hours. The minimum dilution concentration without bacterial growth was the minimum inhibitory concentration, i.e., MIC. All experiments were repeated 3 times in parallel.

[0080] Combination Index (CI): CI is an important parameter to measure whether the combination of compounds has synergistic or antagonistic effects. The Chou-Talalay model is a commonly used pharmacological model used to evaluate the synergistic and antagonistic effects of compound combinations during the onset of action. The calculation results of CI are shown in the following table. If CI < 1, it means there is a synergistic effect; if CI = 1, it means there is no interaction; if CI > 1, it means there is an antagonistic effect.

[0081] The synergy index (CI) was calculated according to the Chou-Talalay formula:

[0082] Among them C A , C B Indicates the inhibitory effect observed at the corresponding concentration when each component is compounded into a composite; C a , C b They are the concentrations corresponding to the antibacterial effects of components A and B when acting alone; the experimental results are shown in Table 6.

[0083] Table 6:

[0084] Experimental Example 4 The inhibition zone experiment of Propionibacterium acnes was used to demonstrate the synergistic anti-Propionibacterium acnes effect of cryptotanshinone encapsulated by glycyrrhizic acid.

[0085] Experimental method: The glycyrrhizic acid-encapsulated cryptotanshinone nano-micelles of the embodiment and the comparative example were appropriately diluted with deionized water to prepare a MIC liquid concentration of more than 2 orders of magnitude (100 times) for use; Propionibacterium acnes was cultured on an RCM culture medium plate, a small round paper disc (diameter 6.0 mm) was planted on the plate, and 10 μL of the sample to be tested, 10 μL of deionized water (blank control), 10 μL of glycyrrhizic acid solution, and 10 μL of cryptotanshinone solution (diluted with anhydrous ethanol) were dripped on the paper disc; the diameter of the inhibition zone was measured after incubation in an incubator at 37°C for 24 h and 48 h, respectively. The experiment was performed in parallel 3 times. The experimental results are shown in Table 7 and Figure 4 .

[0086] Figure 4 This is a graph showing the experimental results of the Propionibacterium acnes inhibition zone of Example 1 of the present invention and Comparative Example 2.

[0087] Table 7 Glycyrrhizic acid-cryptotanshinone nanomicelles antibacterial zone experimental results:

[0088] As can be seen from the above table, glycyrrhizic acid and cryptotanshinone both have a certain inhibitory effect on Propionibacterium acnes. In addition, in addition to being a carrier for cryptotanshinone, when glycyrrhizic acid is used in combination, the MIC of the micelle is lower than the MIC of single application. According to the CI value results, micelle administration shows a significant synergistic effect on Propionibacterium acnes, proving that encapsulating cryptotanshinone with glycyrrhizic acid has an enhanced inhibitory effect on Propionibacterium acnes, thereby reducing the relative amount of cryptotanshinone. At the same time, the solvent of the glycyrrhizic acid-cryptotanshinone micelle is water, which has good prospects for future cosmetic formulations applied to water systems.

[0089] Experimental Example 5 Evaluation of the anti-acne efficacy of the invention from an in vitro acne animal mouse model Experimental methods: 1. Evaluation of antibacterial and anti-acne effects in vivo: 1.1 Animal grouping and modeling: In this experiment, 4-6 week-old BALB / c male mice were injected intradermally with P. acnes to establish a mouse back acne model. The specific operation method is to shave the hair on the back of the mouse after 3 days of adaptive feeding in a suitable environment, and then use a small amount of depilatory cream to stay for 3 minutes for depilation, and then slowly rinse the depilatory area with an appropriate amount of deionized water. One day later, except for the blank group, the freshly prepared 2.0×10 9 The P. acnes solution with CFU / mL was intradermally injected into the back skin of nude mice (0.05mL / 10g body weight), and the model was established for 7 days. The blank group was injected with sterile saline at the same position as a control.

[0090] The experimental animals were then divided into 5 groups, namely, model group, positive control group (cryptotanshinone ointment), cryptotanshinone group (applied with the product of comparative example 2), glycyrrhizic acid group (applied with the product of comparative example 1), cryptotanshinone-glycyrrhizic acid micelle group (applied with nanomicelles of Example 1), with 10 animals in each group.

[0091] 1.2 Animal administration: Sample preparation: Prepare the product of Comparative Example 2, the product of Comparative Example 1, and the glycyrrhizic acid-encapsulated cryptotanshinone nano-micelles of Example 1 as samples respectively and set aside.

[0092] Administration: On the second day after the modeling, the above sample was applied to the acne site of mice in each treatment group at 0.05g / 10g body weight. The model group did not receive any treatment, and the positive control group was applied with an equal dose of kesocryptone ointment. The administration lasted for 7 days. The swelling of the acne site was observed every day, the weight was weighed, the back of the mouse was photographed, and the length and width of the swollen site were measured with a vernier caliper. The skin swelling volume was calculated by the following formula. After the administration, the mice were decapitated and the back skin of the mice was peeled off for subsequent experiments.

[0093] Swelling volume = length × width 2 ÷2.

[0094] 1.3 Calculation of in vivo antibacterial efficiency: After the administration, a sterile 8mm skin biopsy punch was used to obtain acne skin samples. The skin samples were homogenized in 2ml of PBS, and 0.1ml of the homogenate was diluted 10 times and smeared on the RCM solid plate. It was cultured at 37°C for 48h (anaerobic environment), and the number of colonies formed in each plate was recorded. According to the dilution multiple, the total number of live bacteria contained in each milliliter of the original sample was calculated, and the in vivo antibacterial efficiency of each group was calculated by the number of live bacteria in the skin of each treatment group / the number of live bacteria in the skin of the model group.

[0095] 1.4 Preparation of skin pathology sections: After the administration, the skin of the acne area of ​​each group of mice was peeled off and fixed in 4% paraformaldehyde. After dehydration, soaking and embedding, it was cut into 4 μm thick slices and stained with hematoxylin and eosin. The specific steps are shown in Table 8.

[0096] Table 8:

[0097] The experimental results are shown in Figure 5 , Figure 6 .

[0098] Figure 5 This is a local observation picture of the treatment of acne in each test group of the acne mouse model provided in Experimental Example 5 of the present invention.

[0099] Figure 6 This is a graph showing changes in acne swelling volume in each experimental group of the acne mouse model provided in Experimental Example 5 of the present invention after acne treatment.

[0100] From the analysis of the data, it can be seen that the glycyrrhizic acid-cryptotanshinone nanomicelle group can play a role in treating acne. After one week of administration, it can suppress acne to a smaller level and reduce inflammation. There is no obvious change in the acne volume in the blank group that does not receive the drug.

[0101] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A nano-micelle of cryptotanshinone encapsulated with glycyrrhizic acid, characterized in that: The nano micelles are mainly formed by self-assembly of glycyrrhizic acid and cryptotanshinone in water; The glycyrrhizic acid interacts with cryptotanshinone to form nano micelles with glycyrrhizic acid as a carrier and cryptotanshinone encapsulated in the glycyrrhizic acid; The raw materials for preparing the nano micelles include: cryptotanshinone, glycyrrhizic acid and water, and the ratio of cryptotanshinone, glycyrrhizic acid and water is 1g: 10-20g: 800-1200ml; The particle size of the nano micelles is 4-15 nm, and the polydispersity index PDI is 0.235-0.

392.

2. The glycyrrhizic acid-encapsulated cryptotanshinone nanomicelles according to claim 1, characterized in that: The ratio of cryptotanshinone, glycyrrhizic acid and water is 1g:15-20g:800-1200ml.

3. The glycyrrhizic acid-encapsulated cryptotanshinone nano-micelles according to claim 1, characterized in that: The water is purified water.

4. The method for preparing the glycyrrhizic acid-encapsulated cryptotanshinone nanomicelles according to any one of claims 1 to 3, characterized in that: The preparation method comprises: Cryptotanshinone and glycyrrhizic acid are sequentially added to a solvent to be dissolved, and then the solvent is evaporated and recovered to obtain a film; the film is hydrated and dispersed using a film hydration method to obtain water-soluble glycyrrhizic acid-encapsulated cryptotanshinone nano-micelles.

5. The method for preparing the glycyrrhizic acid-encapsulated cryptotanshinone nanomicelles according to claim 4, characterized in that: The solvent includes at least one of anhydrous ethanol, dichloromethane and chloroform.

6. The method for preparing the glycyrrhizic acid-encapsulated cryptotanshinone nano-micelles according to claim 4, characterized in that: The method for evaporating and recovering the solvent is a rotary evaporation method; The parameters of the rotary evaporation method include: a rotary evaporation temperature of 50-60° C., a rotation speed of 80-120 rpm / min, a vacuum degree of 40-100 kPa, and a time of 20-30 min.

7. The method for preparing the glycyrrhizic acid-encapsulated cryptotanshinone nano-micelles according to claim 4, characterized in that: The thin film hydration method comprises: Purified water is added into the film, followed by hydration, dissolution, incubation and ultrasonic dispersion in sequence to obtain water-soluble glycyrrhizic acid-encapsulated cryptotanshinone nano-micelles.

8. The method for preparing the glycyrrhizic acid-encapsulated cryptotanshinone nano-micelles according to claim 7, characterized in that: The hydration and dissolution incubation temperature is 45-80° C. and the time is 30-90 min.

9. The method for preparing the glycyrrhizic acid-encapsulated cryptotanshinone nano-micelles according to claim 8, characterized in that: The hydration and dissolution incubation temperature is 50-60° C. and the time is 50-70 min.

10. The method for preparing the glycyrrhizic acid-encapsulated cryptotanshinone nano-micelles according to claim 7, characterized in that: The frequency of the ultrasonic dispersion is 20-80 kHz, and the time is 10-30 min.

11. The method for preparing the glycyrrhizic acid-encapsulated cryptotanshinone nano-micelles according to claim 10, characterized in that: The frequency of the ultrasonic dispersion is 40-60 kHz, and the time is 10-15 min.

12. Use of the glycyrrhizic acid-encapsulated cryptotanshinone nanomicelles according to any one of claims 1 to 3 in the preparation of acne prevention and treatment products.

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