Nano-micelles of cryptotanshinone encapsulated by glycyrrhizic acid, and preparation method and application thereof

Abstract

The present invention provides a nano-micelle of glycyrrhizic acid encapsulating cryptotanshinone, a preparation method and an application thereof, which relate to the technical field of skin care products. The nano-micelle of glycyrrhizic acid encapsulating cryptotanshinone uses glycyrrhizic acid as a carrier for carrying cryptotanshinone. Glycyrrhizic acid and cryptotanshinone cooperate synergistically to jointly exert the effects of antibacterial, anti-inflammatory and anti-lipid secretion, and have an obvious synergistic effect; at the same time, the preparation of the nano-micelle effectively alleviates the problems that the existing cryptotanshinone has poor water solubility and poor stability and is difficult to be applied to the water-based cosmetic formulation. In addition, through detection and verification, the nano-micelle of glycyrrhizic acid encapsulating cryptotanshinone of the present invention can effectively reduce the toxicity of cryptotanshinone to keratinocytes compared with the single use of cryptotanshinone, and can intervene in various main pathogenesis of acne, and has antibacterial, acne-removing and repair effects.

Description

Technical Field

[0001] The present invention relates to the technical field of skin care products, and particularly to a nano - micelle encapsulating cryptotanshinone with glycyrrhizic acid, its preparation method and application. Background Art

[0002] Acne is a chronic inflammatory skin disease of the pilosebaceous unit, commonly known as acne vulgaris or acne, which is one of the most common skin problems and mostly occurs in areas rich in sebaceous glands such as the face, neck, chest and back.

[0003] Related research shows that the formation of acne is related to excessive sebaceous gland secretion, over - growth of bacteria, pore blockage, endocrine factors, genetics, diet and living 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 follicle. Further excessive lipid secretion and hyperkeratosis at the infundibular part of the hair follicle cause sebum accumulation, leading to an increase in Propionibacterium acnes around the hair follicle. It decomposes triglycerides in sebum to produce free fatty acids, and at the same time chemotactically attracts inflammatory cells and mediators, ultimately inducing and exacerbating the inflammatory response.

[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, its preparation method and application. Cryptotanshinone is added to mPEG activated ester, and a new hydrophilic tanshinone derivative is prepared through steps such as stirring and dialysis, which can solve problems such as excessive sebum secretion, follicular keratinization, pore blockage, and acne - related bacterial infection. Patent application CN117357429A relates to an anti - acne composition and its use method. This cosmetic composition generally includes a cosmetically acceptable carrier and a combination of skin - active ingredients including sulfur, niacinamide, and phenethyl resorcinol, which can be used to treat acne, treat inflammation, and provide anti - aging and other benefits to the skin. However, the preparation method of the above - disclosed tanshinone derivative is too cumbersome, involving continuous stirring for 12 hours and dialysis for 48 hours. At the same time, the above method uses dichloromethane and other ingredients prohibited in cosmetics for preparation, and there may be safety problems such as residual organic solvents.

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

[0006] First, as a lipophilic component, cryptotanshinone has extremely low solubility in water. This makes it difficult for cryptotanshinone to be evenly dispersed and dissolved when preparing aqueous cosmetics, thus affecting the stability and performance of the products. For example, when preparing acne treatment essence or cream, the poor water solubility of cryptotanshinone can lead to layering, precipitation or crystallization in the products, which not only affects the appearance and texture of the products, but may also reduce their acne treatment effects. In patent CN109045109A, the inventors tried to improve the dispersibility of cryptotanshinone in water by adding surfactants. However, although this method can improve the dispersion degree of cryptotanshinone to a certain extent, the use of surfactants may also have potential impacts on the mildness and safety of the products.

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

[0008] In addition, at present, the treatment effects of anti-acne cosmetic products or anti-acne products on the market are limited, and there are side effects such as easy recurrence, skin dryness and desquamation, still unable to fully meet the needs of consumers to improve acne problems.

[0009] In contrast, the glycyrrhizic acid encapsulation technology proposed in this application can not only effectively improve the water solubility of cryptotanshinone, but also maintain the mildness and safety of the products; at the same time, the glycyrrhizic acid encapsulation technology proposed in this application can effectively protect cryptotanshinone from the influence of 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 specifically proposed. Summary of the Invention

[0011] The purpose of the present invention is to provide a nano-micelle of glycyrrhizic acid-encapsulated cryptotanshinone and its preparation method. The nano-micelle uses glycyrrhizic acid as a carrier and encapsulates cryptotanshinone by the thin film dispersion hydration method to obtain the nano-micelle. At the same time, based on the mechanism of acne, through the synergy of the glycyrrhizic acid carrier and cryptotanshinone, the efficacy of cryptotanshinone in treating acne is improved from multiple angles, effectively solving the problems of poor water solubility and stability of cryptotanshinone and its difficulty in being applied to the water-soluble cosmetic formulation system, and further enriching the raw material market for acne treatment cosmetics.

[0012] To achieve the above object of the present invention, the following technical solutions are specifically adopted:

[0013] A nanomicelle of cryptotanshinone encapsulated by glycyrrhizic acid provided by the present invention, wherein the nanomicelle is mainly formed by the self-assembly of glycyrrhizic acid and cryptotanshinone in water;

[0014] Glycyrrhizic acid and cryptotanshinone interact with each other to form a nanomicelle with glycyrrhizic acid as a carrier and cryptotanshinone encapsulated inside glycyrrhizic acid.

[0015] Further, the raw materials for preparing the nanomicelle include: cryptotanshinone, glycyrrhizic acid and water, and the ratio of cryptotanshinone, glycyrrhizic acid and water is 1 g: 10-20 g: 800-1200 ml.

[0016] Further, the ratio of cryptotanshinone, glycyrrhizic acid and water is 1 g: 15-20 g: 800-1200 ml;

[0017] Further, the water is purified water.

[0018] Further, the particle size of the nanomicelle is 4-15 nm, and the polydispersity index PDI is 0.235-0.392.

[0019] A method for preparing a nanomicelle of cryptotanshinone encapsulated by glycyrrhizic acid provided by the present invention, wherein the preparation method includes:

[0020] Cryptotanshinone and glycyrrhizic acid are successively added to a solvent for dissolution, and then the solvent is evaporated and recovered to obtain a film; the film is hydrated and dispersed by the film hydration method to obtain a water-soluble nanomicelle of cryptotanshinone encapsulated by glycyrrhizic acid.

[0021] Further, the solvent includes at least one of anhydrous ethanol, dichloromethane, and chloroform.

[0022] Further, the method for evaporating and recovering the solvent is the rotary evaporation method;

[0023] The parameters of the rotary evaporation method include: the temperature of rotary evaporation is 50-60 °C, the rotation speed is 80-120 rpm / min, the vacuum degree is 40-100 kPa, and the time is 20-30 min.

[0024] Further, the film hydration method includes:

[0025] Purified water is added to the film, and then hydration dissolution incubation and ultrasonic dispersion are successively carried out to obtain a water-soluble nanomicelle of cryptotanshinone encapsulated by glycyrrhizic acid.

[0026] Furthermore, the temperature of the hydration dissolution incubation is 45-80 °C, and the time is 30-90 min;

[0027] Preferably, the temperature of the hydration dissolution incubation is 50-60°C and the time is 50-70 minutes;

[0028] Furthermore, the frequency of the ultrasonic dispersion is 20-80 kHz, and the time is 10-30 min;

[0029] Preferably, the frequency of the ultrasonic dispersion is 40-60 kHz, and the time is 10-15 min.

[0030] The invention provides an application of the glycyrrhizic acid-encapsulated cryptotanshinone nano-micelles in the preparation of acne prevention and treatment products.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] 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.

[0033] 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.

[0034] 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

[0035] 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.

[0036] Figure 1 This is a particle size distribution diagram of the glycyrrhizic acid-encapsulated cryptotanshinone nanomicelles prepared in Example 1 of the present invention;

[0037] Figure 2 This is the infrared spectrum (FT-IR) diagram of the glycyrrhizic acid-coated cryptotanshinone nanomicelles prepared in Example 1 of the present invention;

[0038] Figure 3 This is the infrared spectrum (FT-IR) diagram of the product prepared in Comparative Example 1 of the present invention;

[0039] Figure 4 This is the experimental result diagram of the antibacterial zone of Propionibacterium acnes in Example 1 and Comparative Example 2 provided in Experimental Example 4 of the present invention;

[0040] Figure 5 This is the local observation diagram of treating acne in each test group of the acne mouse model provided in Experimental Example 5 of the present invention;

[0041] Figure 6 This is the diagram of the change in the acne swelling volume of treating acne in each test group of the acne mouse model provided in Experimental Example 5 of the present invention. Detailed implementation manners

[0042] Next, the technical solutions of the present invention will be clearly and completely described 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 those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0043] According to one aspect of the present invention, a nanomicelle of glycyrrhizic acid-coated cryptotanshinone is provided, and the nanomicelle is mainly formed by the self-assembly of glycyrrhizic acid and cryptotanshinone in water;

[0044] The glycyrrhizic acid and cryptotanshinone interact with each other to form a nanomicelle with glycyrrhizic acid as the carrier and cryptotanshinone encapsulated inside the glycyrrhizic acid.

[0045] The nanomicelle of glycyrrhizic acid-coated cryptotanshinone provided by the present invention uses glycyrrhizic acid as the carrier for carrying cryptotanshinone, and through the synergistic effect with cryptotanshinone, it simultaneously exerts the effects of antibacterial, anti-inflammatory, and anti-lipid secretion, effectively alleviating the problems that the existing cryptotanshinone has poor water solubility and stability and is difficult to be applied to the cosmetic formulations in the water system.

[0046] Meanwhile, it should be noted that the carrier glycyrrhizic acid (glycyrrhizicacid) in the present application is an amphiphilic active ingredient derived from the root of traditional Chinese medicine licorice, containing two molecules of glucuronic acid and one molecule of glycyrrhetinic acid, having water solubility and capable of self-assembling with lipophilic components to form nanomicelles, which are also called nanovesicles. In addition, research has confirmed that glycyrrhizic acid is one of the most important active ingredients in licorice, having effects such as antiviral, anti-inflammatory, antioxidant, immunomodulatory, and liver protection.

[0047] Therefore, the present application uses glycyrrhizic acid as a carrier, which can effectively alleviate the problems of poor water solubility and poor stability of cryptotanshinone, and at the same time can exert its own anti-inflammatory effect, thereby giving play to the characteristics and advantages of "combining medicine and adjuvant". In addition, compared with the use of cryptotanshinone alone, the antibacterial, anti-inflammatory, and anti-lipid secretion effects of the nanomicelles of the present invention are all more effective. Through detection and verification, it can reduce the toxicity of cryptotanshinone to keratinocytes, and glycyrrhizic acid can play a synergistic effect with cryptotanshinone while serving as a carrier, and can effectively intervene in various main pathogenesis of acne, and has antibacterial, anti-acne and repair effects.

[0048] In a preferred embodiment of the present invention, the raw materials for preparing the nanomicelles include: cryptotanshinone, glycyrrhizic acid and water, and the ratio of cryptotanshinone, glycyrrhizic acid and water is 1 g: 10-20 g: 800-1200 ml.

[0049] In the above preferred embodiment, the ratio of cryptotanshinone, glycyrrhizic acid and water is 1 g: 15-20 g: 800-1200 ml;

[0050] In a preferred embodiment of the present invention, the water is purified water.

[0051] 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.

[0052] According to one aspect of the present invention, a method for preparing nanomicelles of glycyrrhizic acid-coated cryptotanshinone, the preparation method comprising:

[0053] Adding cryptotanshinone and glycyrrhizic acid to a solvent in sequence for dissolution, and then evaporating and recovering the solvent to obtain a film; using the film hydration method to hydrate and disperse the film to obtain water-soluble nanomicelles of glycyrrhizic acid-coated cryptotanshinone.

[0054] The preparation method of the nanomicelles of glycyrrhizic acid-coated cryptotanshinone provided by the present invention uses glycyrrhizic acid as a carrier, and adopts the film dispersion hydration method to encapsulate cryptotanshinone inside glycyrrhizic acid to form water-soluble nanomicelles of glycyrrhizic acid-coated cryptotanshinone. The above preparation method has the technical advantages of simple processing technology and easy operation.

[0055] In a preferred embodiment of the present invention, the solvent includes at least one of anhydrous ethanol, dichloromethane, and chloroform.

[0056] In a preferred embodiment of the present invention, the method for evaporating and recovering the solvent is rotary evaporation;

[0057] The parameters of the rotary evaporation method include: the temperature of rotary evaporation is 50 - 60 °C, the rotation speed is 80 - 120 rpm / min, the vacuum degree is 40 - 100 kPa, and the time is 20 - 30 min.

[0058] In a preferred embodiment of the present invention, the film hydration method includes:

[0059] Purified water is added to the film, and then hydration dissolution incubation and ultrasonic dispersion are carried out in sequence to obtain water-soluble glycyrrhizic acid-coated cryptotanshinone nanomicelles.

[0060] In the above preferred embodiment, the temperature of the hydration dissolution incubation is 45 - 80 °C, and the time is 30 - 90 min;

[0061] Preferably, the temperature of the hydration dissolution incubation is 50 - 60 °C, and the time is 50 - 70 min;

[0062] In the above preferred embodiment, the frequency of the ultrasonic dispersion is 20 - 80 kHz, and the time is 10 - 30 min;

[0063] Preferably, the frequency of the ultrasonic dispersion is 40 - 60 kHz, and the time is 10 - 15 min.

[0064] Preferably, the glycyrrhizic acid-coated cryptotanshinone nanomicelles and its preparation method include:

[0065] S1: Dissolve cryptotanshinone in a solvent, add glycyrrhizic acid, and if necessary, assist dissolution by ultrasonic, to obtain an orange-red clear and transparent mixture solution;

[0066] S2: Rotate and evaporate the mixture solution to recover the solvent, and disperse it to form a uniform film;

[0067] S3: Add purified water to the dispersed film system, carry out hydration dissolution incubation and ultrasonic dispersion to obtain water-soluble glycyrrhizic acid-coated cryptotanshinone nanomicelles.

[0068] According to one aspect of the present invention, the above-mentioned glycyrrhizic acid-coated cryptotanshinone nanomicelles are used in the preparation of acne prevention and treatment products.

[0069] The glycyrrhizic acid-coated cryptotanshinone nanomicelles provided by the present invention can be widely used in the preparation process of acne prevention and treatment products.

[0070] 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, anti-acne or repair effects;

[0071] And / or, the acne prevention and treatment product is a cosmetic having at least one or more of antibacterial, anti-acne or repair effects.

[0072] The technical solution of the present invention will be further described below in conjunction with embodiments.

[0073] Embodiment 1

[0074] A nano - micelle of cryptotanshinone encapsulated by glycyrrhizic acid and its preparation method. Using glycyrrhizic acid as a carrier, cryptotanshinone is encapsulated by the thin - film dispersion hydration method, which specifically includes the following steps:

[0075] S1: Weigh 10 mg of cryptotanshinone and 150 mg of glycyrrhizic acid precisely, and dissolve them fully with 15 mL of anhydrous ethanol solution to obtain a mixed solution of cryptotanshinone and glycyrrhizic acid, which is a clear orange - red liquid.

[0076] S2: Rotate and evaporate the mixture for 30 min at 70 kPa, a temperature of 55 °C, and a rotation speed of 100 rpm to remove ethanol, and form a uniform thin film on the wall of the egg - shaped flask of the rotary evaporator.

[0077] S3: Add 10 mL of purified water in small amounts and multiple times at 55 °C to completely dissolve the thin film in water, incubate at 55 °C for 60 min with constant - temperature hydration and dissolution, disperse ultrasonically at 50 kHz for 15 min, and filter to finally obtain an orange - red clear solution.

[0078] 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.

[0079] Embodiments 2 - 5

[0080] Except that the ratio of "cryptotanshinone, glycyrrhizic acid and water" in Embodiments 2 - 5 is different from that in Embodiment 1, the rest are the same as Embodiment 1. The ratios of cryptotanshinone, glycyrrhizic acid and water in Embodiments 2 - 5 are shown in Table 1.

[0081] Table 1:

[0082]

[0083] Embodiments 6 - 10

[0084] Except that the rotary evaporation parameters in step S2 of Embodiments 6 - 10 are different from those in Embodiment 1, the rest are the same as Embodiment 1. The rotary evaporation parameters in Embodiments 6 - 10 are shown in Table 2.

[0085] Table 2:

[0086]

[0087] Embodiment 11

[0088] The difference between this example and Example 1 is that the hydration dissolution incubation time in step S3 is 30 min, and the other parts are the same as those in Example 1.

[0089] Example 12

[0090] The difference between this example and Example 1 is that the hydration dissolution incubation time in step S3 is 90 min, and the other parts are the same as those in Example 1.

[0091] Comparative Examples 1 - 5

[0092] Comparative Examples 1 - 5 are the same as Example 1 except that the "composition and ratio of cryptotanshinone, glycyrrhizic acid and water" are different from those in Example 1. For cryptotanshinone, glycyrrhizic acid and water in Comparative Examples 1 - 5, see Table 3.

[0093] Table 3:

[0094]

[0095] Comparative Example 6

[0096] The difference between this comparative example and Example 1 is that the rotation speed of rotary evaporation in step S2 is 30 rpm, and the other parts are the same as those in Example 1.

[0097] Comparative Example 7

[0098] The difference between this comparative example and Example 1 is that the rotary evaporation time in step S2 is 10 min, and the other parts are the same as those in Example 1.

[0099] Comparative Example 8

[0100] The difference between this comparative example and Example 1 is that the hydration dissolution incubation time in step S3 is 10 min, and the other parts are the same as those in Example 1.

[0101] Comparative Example 9

[0102] The difference between this comparative example and Example 1 is that the hydration dissolution incubation temperature in step S3 is 40 °C, and the other parts are the same as those in Example 1.

[0103] Comparative Example 10

[0104] 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 those in Example 1.

[0105] Next, the nano - micelles of glycyrrhizic acid - encapsulated cryptotanshinone prepared by the present invention will be subjected to the following experiments to further elaborate on the advantages of the present invention.

[0106] Experimental Example 1 Particle Size and Zeta Potential Experiment of Glycyrrhizic Acid - Encapsulated Cryptotanshinone Nano - Micelles

[0107] 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.

[0108] Table 4:

[0109]

[0110] Table 5:

[0111]

[0112] 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.

[0113] Figure 1 This is the particle size distribution diagram of the glycyrrhizic acid-encapsulated cryptotanshinone nanomicelles prepared in Example 1 of the present invention.

[0114] 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;

[0115] 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;

[0116] 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);

[0117] 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);

[0118] 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;

[0119] 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;

[0120] In Comparative Example 8, the drug film cannot be completely dissolved in step S3 due to the short hydration and dissolution incubation time;

[0121] In Comparative Example 9, due to the too low temperature of hydration dissolution incubation, step S3 could not completely dissolve the drug film;

[0122] In Comparative Example 10, due to the too short time of ultrasonic dispersion, the particle size distribution was uneven and large (0.609 ± 0.138).

[0123] Experimental Example 2 Fourier transform infrared spectroscopy (FT-IR) characterization of tanshinone cryptotanshinone-loaded glycyrrhizic acid nanomicelles

[0124] Experimental method: The Fourier transform infrared spectroscopy (FT-IR) instrument was used to measure the infrared absorption spectrum of tanshinone cryptotanshinone-loaded glycyrrhizic acid nanomicelles.

[0125] Specifically, after mixing the samples prepared in Example 1, Example 2, Example 3, Comparative Example 1, Comparative Example 2, and Comparative Example 3 with 200 mg of KBr respectively, they were ground evenly, placed in an infrared tablet pressing mold and pressed into thin slices. Different samples were collected through 32 scans, the wavelength was 4000 - 400 cm-1, and the resolution was 2. The experimental results can be seen in Figure 2 and Figure 3 .

[0126] Figure 2 This is the infrared spectrum (FT-IR) diagram of tanshinone cryptotanshinone-loaded glycyrrhizic acid nanomicelles prepared in Example 1 of the present invention.

[0127] Figure 3 This is the infrared spectrum (FT-IR) diagram of the product prepared in Comparative Example 1 of the present invention.

[0128] Comparing the results of Example 1 and Comparative Example 1, it was found that the infrared absorption characteristic peak of tanshinone cryptotanshinone-loaded glycyrrhizic acid nanomicelles at 3442.61 cm-1 was red-shifted to 3423.20 cm -1 and the transmittance was significantly increased. The change of the FTIR characteristic absorption peak proved that tanshinone cryptotanshinone was successfully encapsulated by glycyrrhizic acid.

[0129] Experimental Example 3 Proving the synergistic anti - Propionibacterium acnes effect of tanshinone cryptotanshinone encapsulated by glycyrrhizic acid through the MIC of Propionibacterium acnes

[0130] Experimental method:

[0131] (1) Preparation of samples to be tested: Samples were prepared by using the experimental methods of Example 1, Comparative Example 1, and Comparative Example 2 respectively.

[0132] (2)Activation and preparation of bacterial strains: The Propionibacterium acnes (ATCC 11827) strain was purchased from the Guangdong Provincial Microbial Culture Collection Center (GDMCC). Weighed 11.4 g of Reinforced Clostridial Medium (RCM) and 3.6 g of agar powder into a clean Erlenmeyer flask, added 300 ml of distilled water, heated and dissolved it. After complete dissolution, autoclaved at 121 °C for 30 min for later use. Inoculated the Propionibacterium acnes strain onto the RCM medium, cultured at 37 °C for 20 - 28 hours for activation. After transferring to the third generation, the experiment began.

[0133] (3)MIC experiment: Inoculated the preserved Propionibacterium acnes bacteria onto the Reinforced Clostridial Medium (RCM), cultured at 37 °C for activation. Picked a small amount of bacterial colonies, diluted them with sterile normal saline to 0.5 McFarland units to prepare the inoculation bacterial suspension, sealed it and stored it refrigerated for later use. Detected the MIC value of the plant compound for inhibiting Propionibacterium acnes composition extract in the preparation examples and comparative preparation examples with 2 mL EP tubes for each drug concentration detected in each row.

[0134] Conducted the experiment using the microdilution method. Added 500 μL of medium to all 2 mL EP tubes, then added 500 μL of the plant compound for inhibiting Propionibacterium acnes composition liquid medicine to the first well. Drew 500 μL of the mixed solution from the first well and added it to the second well, and so on until the 12th well, obtaining drugs diluted 2, 4, 8, 16, 32, 64, 128, 256, 512, 1024, 2048, 4096 times. Discarded 500 μL of the liquid medicine from the 12th well, and then added 500 μL of the inoculation bacterial suspension to each of the wells 1 - 13. Among them, well 13 was the growth group, and well 14 was the blank group. Observed the growth of Propionibacterium acnes after culturing at 37 °C under constant temperature conditions for 24 hours. The lowest dilution concentration without bacterial growth was the minimum inhibitory concentration, that is, MIC. All experiments were repeated in parallel 3 times.

[0135] Combination Index (CI): CI is an important parameter to measure whether the combination of compositions has a synergistic effect or an antagonistic effect. The Chou - Talalay model is a commonly used pharmacological model for evaluating the synergistic and antagonistic effects of compound combinations during the onset process. The calculation results of CI are shown in the following table. If CI < 1, it indicates the existence of a synergistic effect; if CI = 1, it indicates the absence of interaction; if CI > 1, it indicates the existence of an antagonistic effect.

[0136] Calculated the combination index (CI) according to the Chou - Talalay formula, and the formula is:

[0137]

[0138] Among them, C A , C B represents the inhibitory effect observed at the corresponding concentration when each component is compounded into a composition; C a , C b are the concentrations corresponding to the antibacterial effects of components A and B acting alone respectively; the experimental results are shown in Table 6 below.

[0139] Table 6:

[0140]

[0141] Experimental Example 4 demonstrated the synergistic anti - Propionibacterium acnes effect of cryptotanshinone encapsulated by glycyrrhizic acid through the antibacterial zone experiment of Propionibacterium acnes.

[0142] Experimental method: Appropriately dilute the glycyrrhizic acid - encapsulated cryptotanshinone nanomicelles of the examples and comparative examples with deionized water respectively to prepare MIC solutions with concentrations of more than 2 orders of magnitude (100 - fold), and set aside; culture Propionibacterium acnes on an RCM medium plate, place small round filter papers (with a diameter of 6.0 mm) on the plate, and drop 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 absolute ethanol) on the papers respectively; measure the diameter of the antibacterial zone after incubating in an incubator at 37 °C for 24 h and 48 h respectively. The experiment was carried out in parallel 3 times, and the experimental results are shown in Table 7 below and Figure 4 .

[0143] Figure 4 This is the experimental result graph of the antibacterial zone of Propionibacterium acnes for Example 1 and Comparative Example 2 of the present invention.

[0144] Table 7 Experimental results of the antibacterial zone of glycyrrhizic acid - cryptotanshinone nanomicelles:

[0145]

[0146] As can be seen from the above table, both glycyrrhizic acid and cryptotanshinone have certain inhibitory effects on Propionibacterium acnes. In addition, in addition to being a carrier for carrying cryptotanshinone, when used in combination, the MIC of the micelles is lower than that of the individual use. According to the CI value results, the micelle administration shows an obvious synergistic effect on Propionibacterium acnes, which proves that encapsulating cryptotanshinone with glycyrrhizic acid can enhance the inhibitory effect on Propionibacterium acnes, thereby reducing the relative dosage of cryptotanshinone. At the same time, the solvent of the glycyrrhizic acid - cryptotanshinone micelle is water, which has good prospects for future application in cosmetic formulations for water - based systems.

[0147] Experimental Example 5 evaluated the anti - acne efficacy of the present invention from an in vitro acne animal mouse model

[0148] Experimental method:

[0149] 1. Evaluation of antibacterial and anti - acne effects in vivo:

[0150] 1.1 Animal grouping and model establishment:

[0151] In this experiment, a mouse back acne model was established by intradermal injection of P.acnes into the back skin of 4 - 6 - week - old male BALB / c mice. The specific operation method was as follows: After adaptively feeding for 3 days in a suitable environment, the hair on the back of the mice was shaved off, and a small amount of depilatory cream was left for 3 min for hair removal, and then the depilated area was slowly rinsed with an appropriate amount of deionized water. One day later, except for the blank group, a freshly prepared P.acnes bacterial solution of 2.0×10 9 CFU / mL was intradermally injected into the back skin of nude mice (0.05 mL / 10 g body weight), and the model establishment continued for 7 days. The blank group was injected with sterile normal saline at the same position as a control.

[0152] The experimental animals were randomly divided into 5 groups, namely the model group, the positive control group (Kezuoyintong ointment), the cryptotanshinone group (coating the product of Comparative Example 2), the glycyrrhizic acid group (coating the product of Comparative Example 1), and the cryptotanshinone - glycyrrhizic acid micelle group (coating the nano - micelles of Example 1), with 10 animals in each group.

[0153] 1.2 Animal administration:

[0154] Sample preparation: The products of Comparative Example 2, Comparative Example 1, and the cryptotanshinone - loaded glycyrrhizic acid nano - micelles of Example 1 were respectively prepared as samples for standby.

[0155] Administration: On the second day after the model establishment, the above samples were applied to the acne sites of the mice in each treatment group at a dose of 0.05 g / 10 g body weight. The model group was not treated. The positive control group was applied with an equal dose of Kezuoyintong ointment. The administration continued for 7 days. The swelling of the acne sites was observed every day, the mice were weighed, the back of the mice was photographed, and the length and width of the swollen area were measured with a vernier caliper. The skin swelling volume was calculated by the following formula. After the administration was completed, the mice were sacrificed by cervical dislocation, and the back skin of the mice was peeled off for subsequent experiments.

[0156] Swelling volume = length × width 2 ÷2.

[0157] 1.3 Calculation of in - vivo antibacterial efficiency:

[0158] After the administration was completed, acne skin samples were obtained using a sterile 8-mm skin biopsy punch. The skin samples were homogenized in 2 ml of PBS. After diluting the homogenate 10-fold, 0.1 ml was taken and spread on an RCM solid plate, and cultured at 37 °C for 48 h (anaerobic environment). The number of colonies formed in each petri dish was recorded, and based on the dilution factor, the total number of viable bacteria contained in each milliliter of the original sample was calculated. The in vivo antibacterial efficiency of each group was calculated as the number of viable bacteria on the skin in the treatment group / the number of viable bacteria on the skin in the model group.

[0159] 1.4 Preparation of skin pathological sections:

[0160] After the administration was completed, the acne skin of each group of mice was dissected and fixed in 4% paraformaldehyde. After dehydration, immersion, and embedding, it was cut into thin slices with a thickness of 4 μm, stained with hematoxylin and eosin, and the specific steps are shown in Table 8.

[0161] Table 8:

[0162]

[0163] The experimental results are shown in Figure 5 、 Figure 6 。

[0164] Figure 5 are the local observation diagrams of the treatment of acne in each test group of the acne mouse model provided in Experimental Example 5 of the present invention.

[0165] Figure 6 are the diagrams of the change in the acne swelling volume of the treatment of acne in each test group of the acne mouse model provided in Experimental Example 5 of the present invention.

[0166] Analyzing the data results, it can be seen that the glycyrrhizic acid-cryptotanshinone nanomicelle group can play a role in treating acne. After one week of administration, acne can be inhibited to a relatively small level and the inflammatory phenomenon is alleviated. There is no obvious change in the volume of acne in the blank group without administration.

[0167] 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 foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing nano-micelles of cryptotanshinone encapsulated with glycyrrhizic acid, characterized in that: The preparation method comprises: Cryptotanshinone and glycyrrhizic acid are sequentially added to a solvent to dissolve, and then the solvent is evaporated and recovered to obtain a film; the film is hydrated and dispersed by a film hydration method to obtain water-soluble glycyrrhizic acid-encapsulated cryptotanshinone nano-micelles; The raw materials for preparing the nano micelles include: cryptotanshinone, glycyrrhizic acid and purified water, and the ratio of cryptotanshinone, glycyrrhizic acid and purified water is 1g: 10-15g: 800-1200ml; The particle size of the nanomicelles is 4-15 nm, and the polydispersity index PDI is 0.235-0.392; The method for evaporating and recovering the solvent is a rotary evaporation method, and the parameters of the rotary evaporation method include: a rotary evaporation temperature of 50-60°C, a rotation speed of 80-120 rpm, a vacuum degree of 40-100 kPa, and a time of 30 min; The film hydration method comprises: adding purified water to the film, followed by sequentially performing hydration, dissolution, incubation, and ultrasonic dispersion to obtain water-soluble glycyrrhizic acid-encapsulated cryptotanshinone nano-micelles; The frequency of the ultrasonic dispersion is 20-80 kHz, and the time is 10-30 min; The hydration and dissolution incubation temperature is 50-60° C. and the time is 50-70 min.

2. The method for preparing the glycyrrhizic acid-encapsulated cryptotanshinone nano-micelles according to claim 1, characterized in that: The solvent includes at least one of anhydrous ethanol, dichloromethane and chloroform.

3. The method for preparing the glycyrrhizic acid-encapsulated cryptotanshinone nano-micelles according to claim 1, characterized in that: The frequency of the ultrasonic dispersion is 40-60 kHz, and the time is 10-15 min.

4. Use of the glycyrrhizic acid-encapsulated cryptotanshinone nanomicelles obtained by the method for preparing 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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