Application of ellagic acid nanoparticles in preparation of external preparation with noninvasive deep skin drug delivery function

Abstract

The invention provides ellagic acid nanoparticles which can solve the problems of extremely low solubility, transdermal absorption performance and deep skin delivery performance of the ellagic acid nanoparticles without adding a large number of auxiliary materials and a complex preparation process. The compound is used for preparing medicines, cosmetics and daily chemical products with one or more effects of whitening, removing freckles, resisting oxidation, resisting aging, scavenging free radicals, removing wrinkles, blocking sun, enhancing skin elasticity, reducing pigmentation, preserving moisture, resisting bacteria, resisting viruses, treating alopecia, treating white hair, degreasing skin, treating melanoma, repairing scars and healing wounds. The effect of permeating the medicine into the deep layer of the skin is shown, and the medicine can reach and target the fascia layer of the skin at the deepest. The improvement of the deep delivery performance is related to the obvious enhancement of the solubility and is also related to the nanocrystallization property, the development prospect of the ellagic acid nanoparticles in the external preparation, especially the external preparation for intradermal deep drug delivery, and the deep delivery does not depend on devices such as a microneedle and is a completely non-invasive delivery method.

Description

Technical Field

[0001] The invention belongs to the field of external preparation raw materials, cosmetic raw materials and pharmaceutical raw materials, and specifically relates to the application of ellagic acid nanoparticles in the preparation of external preparations with non-invasive deep skin drug delivery function. Technical Background

[0002] Ellagic acid (EA), also known as retrogallic acid, is a plant polyphenol with CAS number 476-66-4 and molecular formula C 14 H6O8, molecular weight 302.19Da, melting point>360℃; characteristic wavelengths of ultraviolet absorption are 254nm and 366nm, insoluble in ethers, slightly soluble in water, alcohol, dimethyl sulfoxide, soluble in pyridine and alkaline solutions. It is widely found in various fruits, nuts and other plants, such as grapes, pomegranates, apples, cherries, raspberries, strawberries, blueberries, tara, walnuts, and Euryale ferox, etc., and has a wide range of effects, such as anti-inflammatory, antibacterial, antiviral, especially antioxidant, anti-aging, whitening, and astringent. Ellagic acid has been applied to many types of cosmetics such as whitening and anti-aging due to its high safety, mild effect on the skin and significant efficacy. Functional cosmetics made with ellagic acid as raw material have great research and development value and market prospects. Ellagic acid has multiple functions in skin care, such as anti-oxidation, whitening and freckle removal, and refreshing skin. As an antioxidant, ellagic acid can effectively remove free radicals, thereby alleviating the problem of skin aging. More specifically, ellagic acid has the effects of whitening, removing spots and wrinkles. It is a natural elastase inhibitor that effectively inhibits the activity of elastase, thereby enhancing the elasticity of the skin, reducing wrinkles and pigmentation, and delaying skin aging. As a moisturizer and antibacterial agent, ellagic acid has a good inhibitory effect on a variety of bacteria and viruses, protecting the skin from bacterial invasion. Ellagic acid inhibits the growth of microorganisms by binding to enzymes involved in the life activities of microorganisms and changing their molecular structures. In addition, after ellagic acid acts on bacterial cells, it can destroy the integrity of the cell wall, increase the permeability of the cell membrane, cause the leakage of metal ions and proteins, and make the cell metabolism system disorder, thereby playing an antibacterial effect. Ellagic acid can be used in the preparation of sunscreen products, and has relevant reports on the treatment of hair loss, treatment of white hair, melanoma, scar repair, wound healing, etc.

[0003] Ellagic acid belongs to Class IV of the Biopharmaceutical Classification System (BCS), i.e., low solubility and low permeability drugs. High lattice force (melting point 360°C) is the root cause of the poor solubility of ellagic acid. The application of EA is seriously hindered by its extremely low solubility (<10μg / mL in water). In the previous study, the inventor used ultrasonic packaging to make ellagic acid self-assemble to form nanoparticles (EANPs), which significantly improved the solubility of EA (>120 times). As a result, two Chinese invention patents were applied for and one academic paper was published, hereinafter referred to as "Patent 1", "Patent 2", and "Article 1":

[0004] Patent 1: Gu Wei; Cheng Jianming; Wang Yiwei; Qi Shuyang; Ji Jing. A nanosuspension of ellagic acid with high solubility and antioxidant activity and its application in oral, topical preparations and cosmetics [P]. Jiangsu Province: CN202211465349.3, 20221122);

[0005] Patent 2: Gu Wei; Chen Jun; Yang Kangli; Qian Yuerong; Tong Huangjin; Hu Qinglian; Li Feng; Zhang Shunan; Xu Fei; Zhao Hongyu. A nanosuspension of ellagic acid and its application in oral, topical preparations and cosmetics [P]. Jiangsu Province: CN202111461097.2.;

[0006] Article 1: Wei Gu, Ruolin Kong, Shuyang Qi, Xuxi Cheng, Xuyi Cai, Ziyun Zhou, Shunan Zhang, Hongyu Zhao, Jinyun Song, Qinglian Hu, Huiwen Yu, Huangjin Tong*, Yiwei Wang*, Tulin Lu*. Sono-assembly of ellagic acid into nanostructures significantly enhances aqueous solubility and bioavailability[J]. Food Chemistry. 2024, 442: 138485. doi: 10.1016 / j.foodchem.2024.138485. PubMed PMID: 38278106. Summary of the invention

[0007] The invention provides an application of ellagic acid nanoparticles in preparing an external preparation with a non-invasive deep skin drug delivery function, wherein the ellagic acid nanoparticles are prepared by the following method: ellagic acid powder is added to a preparation solvent and prepared by an ultrasonic process; the preparation solvent is pure water or an organic solvent or a mixed solution of an organic solvent and water, and the organic solvent is selected from one or more of ethanol, propylene glycol, butylene glycol, pentanediol, and glycerol; the ultrasonic process conditions are: ellagic acid feed concentration ≥1.0 mg / mL, ultrasonic power ≥100 w, ultrasonic frequency ≥20 kHz, ultrasonic temperature ≥30°C, and ultrasonic time ≥5 min; the obtained suspension is subjected to centrifugation, filtration, sedimentation and other methods to remove solid parts, and the supernatant is taken to obtain the ellagic acid nanoparticles.

[0008] As an improvement, in the ultrasonic process conditions, the ultrasonic temperature is ≥ 35°C, more preferably 45°C.

[0009] As another improvement, the ellagic acid feed concentration in the ultrasonic process condition is ≥2.5 mg / mL, more preferably 20.0 mg / mL.

[0010] As another improvement, the preparation solvent is water, or a mixed solution of an organic solvent and water, wherein the ratio of the organic solvent to water is preferably 0:1-3:1 by volume.

[0011] As an improvement, the medicine, cosmetic, and daily chemical product are tinctures, gels, ointments, creams, patches, plasters, foams, lotions, films, and patches, and foams are most preferred.

[0012] As a further improvement, the medicine, cosmetics, and daily chemical products are foaming agents, and their preparation method is as follows: ellagic acid nanoparticles are prepared into a dispersion using an ultrasonic disperser; a surfactant is weighed and added to the dispersion, and a moisturizer and a stabilizer are added at the same time, and the mixture is completely and evenly mixed; triethanolamine is added dropwise to neutralize the system until the pH value reaches 6.5-7.0, so as to form a stable drug-containing liquid foam matrix; the prepared drug-containing liquid foam matrix is ​​loaded into a foam pump bottle, and dispersed by a foam pump to form a stable foam, so as to obtain an ellagic acid nanoparticle foaming agent.

[0013] As a further improvement, the topical preparation has one or more of the functions of whitening, removing freckles, anti-oxidation, anti-aging, scavenging free radicals, removing wrinkles, sun protection, enhancing skin elasticity, reducing pigmentation, moisturizing, antibacterial, antiviral, treating hair loss, treating gray hair, removing skin grease, treating melanoma, scar repair, and wound healing.

[0014] Beneficial Effects

[0015] The present invention provides a kind of ellagic acid nanoparticles, without adding a large amount of auxiliary materials, without having to pass through a complicated preparation process, that is, the problem of extremely low solubility, transdermal absorption performance and deep skin delivery performance of ellagic acid can be solved. It is used to prepare medicines, cosmetics and daily chemicals with one or more effects of preventing or treating whitening, freckle removal, anti-oxidation, anti-aging, free radical scavenging, wrinkle removal, sun protection, enhancing skin elasticity, reducing pigmentation, moisturizing, antibacterial, antiviral, treating hair loss, treating white hair, skin degreasing, treating melanoma and scar repairing functions, showing the effect of deep penetration into the skin, the deepest can directly reach and target the skin fascia layer. The improvement of deep delivery performance is not only related to the significant enhancement of its solubility, but also related to its own nano-nized properties, showing the development prospects of ellagic acid nanoparticles in the field of external preparations, especially deep intradermal delivery of active substances for external preparations. At the same time, this deep delivery does not rely on devices such as micro needles, and is a method of completely non-invasive delivery. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The present invention will be further described below in conjunction with the accompanying drawings:

[0017] Figure 1 :The cumulative transdermal amount of ellagic acid in different test solutions changes with time in the in vitro transdermal experiment, *P<0.5.

[0018] Figure 2 :EANPs foaming agent.

[0019] Figure 3 : Skin fluorescence microscopy and skin HE staining microscopy. DETAILED DESCRIPTION

[0020] The technical solution of the present invention is further described below in conjunction with specific examples, which is not a limitation of the present technical solution. Ethanol, propylene glycol, butylene glycol, pentanediol and other diols and glycerol are commonly used solvents, moisturizers, penetration enhancers or preservatives in topical preparations or oral preparations. The most commonly used 1,3-propylene glycol, 1,3-butylene glycol and 1,2-pentanediol are selected for testing in the following examples, but are not used as limitations of the present technical solution. In addition, the "ellagic acid nanoparticles" in this patent include the "ellagic acid nanosuspension" in "Patent 1", the "ellagic acid nanosuspension" in "Patent 2", and the "Ellagic Acid Nanostructures" in Article 1.

[0021] Example 1 Preparation of Ellagic Acid Nanoparticles (EANPs)

[0022] Ellagic acid (EA) powder is added to the preparation solvent and prepared by ultrasonic process; the ultrasonic process conditions are: ellagic acid feed concentration ≥ 1.0 mg / mL, ultrasonic power ≥ 100w, ultrasonic frequency ≥ 20kHz, the obtained suspension is centrifuged, filtered, sedimented and other methods to remove the solid part, and the supernatant is taken to obtain EANPs. The preparation method can be optimized from the following aspects: the preparation solvent is pure water or an organic solvent or a mixed solution of an organic solvent and water, preferably water, the organic solvent is preferably one or more of ethanol, propylene glycol, butylene glycol, pentanediol, and glycerol, and the ratio of organic solvent to water in the mixed solution of organic solvent and water is preferably 0: 1-3: 1; the ultrasonic temperature in the ultrasonic process conditions is ≥ 30°C, more preferably ≥ 35°C, and most preferably 45°C; the ultrasonic time in the ultrasonic process conditions is ≥ 5min; the ellagic acid feed concentration is preferably ≥ 2.5 mg / mL, and most preferably 20.0 mg / mL.

[0023] Example 2 Determination of ellagic acid concentration by ultraviolet spectrophotometry (refer to Example 3 in "Patent 2")

[0024] Establishment of standard curve: accurately weigh 2.5 mg of ellagic acid standard in a 50 mL volumetric flask and dilute with methanol to prepare a 50 μg / mL standard solution for standby use. Then dilute with ethanol to obtain 12.5, 10, 6.25, 5, 3.125, 1.5625, 0.78125 μg / mL ellagic acid standard solutions, measure the absorbance value under ultraviolet wavelength of 254 nm, draw a standard curve with the mass concentration of ellagic acid standard solution as the abscissa (X) and the corresponding absorbance value (Y) as the ordinate, and perform linear regression on the standard curve to obtain the regression equation: Y = 0.0933X-0.0027, correlation coefficient R 2 =0.9999, indicating that ellagic acid has a good linear relationship in the range of 0.78125-12.5 μg / mL.

[0025] Example 3 Preparation and Characterization of EANPs

[0026] Preparation of EA NPs: Take an appropriate amount of ellagic acid, prepare it into a 20mg / mL suspension with ultrapure water, shake and mix, place it in an ultrasonic cleaner and ultrasonicate it at 45℃ for 1h, then use an ultrasonic cell disruptor to ultrasonicate it for 2min, place the probe 3cm below the liquid in an ice water bath, the condition is 50% power (900W), ultrasonicate is on for 2s and off for 2s. After the ultrasonication is completed, place it in a 50mL centrifuge tube, centrifuge it at 4000rpm for 16min, take the supernatant part, and obtain the EA NPs suspension. Freeze-dry it for 24h to obtain the EA NPs freeze-dried powder.

[0027] Characterization of EA NPs: ① Investigation of EA concentration: The concentration of ellagic acid was determined by the method in Example 2, and the EA content in the prepared EA NPs was 86.96±1.12%. ② Investigation of the equilibrium solubility of EA NPs in water: 20.0 mg EA and EA NPs freeze-dried powder were added to a 10 mL centrifuge tube, and then 4 mL ultrapure water was added. After sealing with a sealing film, it was placed in a 37°C constant temperature water bath for 24 hours, and 3 copies were paralleled. Stop shaking, take the supernatant after free sedimentation for 48 hours, and immediately add methanol to dilute it to the linear range of the standard curve after separation. The absorbance at 254 nm was measured by an ultraviolet spectrophotometer, and the total concentration of EA was substituted into the standard curve to calculate the equilibrium solubility. The results are shown in Table 1. The equilibrium solubility of EA NPs after acoustic cavitation treatment reached 1.64 mg / mL, which was significantly higher than that of free EA.

[0028] Table 1 EA content in EANPs and the equilibrium solubility of EA and EANPs in water (n=3)

[0029]

[0030] Note: Compared with EA, *P<0.05, **P<0.01, ***P<0.001; ND means “not detected”.

[0031] ③ Determination of particle size, potential, and PDI After EA NPs were reconstituted and diluted with ultrapure water, the particle size, PDI, and ζ-potential of the EA NPs suspension were measured using a particle size analyzer. According to Table 2, the particle size of the nanoparticles in the EA NPs suspension is about 270 nm, the PDI is about 0.17, and the particle size is relatively uniform. The ζ potential is -31.6±2.9 mV, which has good stability.

[0032] Table 2 Particle size, PDI, ζ-potential measurement results (n=3)

[0033]

[0034] Example 4 Preparation of EA saturated aqueous solution (EA is in free state at this time)

[0035] The EA saturated aqueous solution was prepared as shown in Table 3.

[0036] Table 3 Ellagic acid content in each group

[0037]

[0038] Note: The EA contents in the table are all data obtained by testing using the equilibrium solubility determination method in Example 3.

[0039] Example 5 In vitro transdermal test

[0040] HPLC method for determination of ellagic acid content (refer to Example 11 in "Patent 2"): ① Chromatographic conditions: The chromatographic column was Hedera C18 column (250 mm × 4.6 mm, 5 μm), and the mobile phase was acetonitrile-0.2% phosphoric acid aqueous solution (V 乙腈 :V 磷酸 =20:80), flow rate 1.0mL / min, detection wavelength 254nm, column temperature 30℃, injection volume 10μL, spectrum acquisition time 15min. ②Linear relationship investigation Take the reference solution to make a 100μg / mL mother solution, dilute it with methanol to 100, 50, 25, 12.5, 6.25, 3.125, 1.5625, 0.78125, 0.390625, 0.1953125μg / mL, inject it into HPLC, and use the mass concentration of ellagic acid reference solution as the horizontal axis (X) and the corresponding peak area as the vertical axis to establish a standard curve (Y), which is Y=1.5937X+0.4033, R 2 =0.9992, and the linear range is 0.1953125~100μg / mL.

[0041] The abdominal skin of rats was used as the transdermal barrier in the in vitro transdermal experiment. Before the experiment, the abdominal skin of healthy rats was cut, cleaned with saline to remove surface residues, and then cut into appropriate sizes to ensure that the skin was intact. The skin stratum corneum was fixed upward between the test cell and the receiving cell of the Franz diffusion cell. The test solution was added to the test cell. The test solution was divided into three groups: ellagic acid nanoparticles, free ellagic acid solution with the same ellagic acid concentration as the nanoparticles, and ellagic acid saturated aqueous solution. The volume of each group was 1 mL to ensure that the reagent covered the entire skin surface. 10 mL of pH 7.4 phosphate buffer was added to the receiving cell as the receiving solution, and its temperature was maintained at 37 ± 0.5 ° C. At the same time, a magnetic stirrer was used to continuously stir to maintain the uniformity of the solution and the stability of mass transfer. The experiment was divided into multiple time points to collect receiving solution samples, including 0, 4, 8, 12, 16, 20 and 24 hours. After each 200 μL sample was collected, an equal amount of fresh phosphate buffer was immediately added. The samples were collected and the concentration of ellagic acid was determined by high performance liquid chromatography (HPLC) to calculate the cumulative amount of ellagic acid that penetrated the skin and entered the receptor fluid per unit time. Figure 1 It can be seen that the transdermal performance of EANPs (Example 3) is significantly better than that of the EA saturated aqueous solution group (Example 4).

[0042] Example 6 Preparation of gel

[0043] First, weigh carbomer 940 and add it to an appropriate amount of pure water, slowly add it while stirring until it is completely swollen to form a uniform basic solution, and let it stand overnight to ensure sufficient swelling; add glycerol and methylparaben to the swollen carbomer basic solution, mix it on a magnetic stirrer to form a preliminary gel matrix, and slowly drop triethanolamine to neutralize it until the pH value reaches 6.5-7.0, so that the gel forms a stable and transparent state. The drug is dispersed with pure water, treated with an ultrasonic disperser (ultrasonic power 50W, ultrasonic time 2 minutes), a stable and uniform dispersion is obtained, and it is slowly added to the prepared gel matrix, and a homogenizer is used to obtain a drug gel after sufficient stirring. The obtained gel has stable properties, a pH value in the range of 6.5-7.0, good smearing performance and penetration-promoting effect, and can be applied to topical treatments in hair loss areas.

[0044] Example 7 Preparation of Foaming Agent

[0045] The drug was dispersed with pure water and treated with an ultrasonic disperser (ultrasonic power 50W, ultrasonic time 2 minutes) to prepare a uniform dispersion. Then, a certain amount of surfactant (such as polysorbate-80) was weighed and added to the dispersion, and a moisturizer (such as glycerol) and a stabilizer (such as carbomer 940) were added to improve the stability of the foam. The mixed solution was placed on a magnetic stirrer and stirred until it was completely uniform. Subsequently, triethanolamine was added dropwise to neutralize the system until the pH value reached 6.5-7.0 to form a stable basic liquid foam matrix. Next, the prepared liquid matrix was loaded into a foam pump bottle and dispersed by a foam pump to form a stable foam, and finally a drug-containing foam was obtained. The obtained foam has a light texture and fine and uniform foam. It can be used for topical treatment of hair loss areas. It is easy to apply and has an excellent user experience. EANPs foams such as Figure 2 shown.

[0046] Example 8 Study on Skin Distribution of Ellagic Acid Nanoparticles

[0047] Male SD rats were taken, their abdominal hair was removed after anesthesia, and they were killed by cervical dislocation. The intact abdominal skin was cut off, the subcutaneous fat tissue was removed, and the rats were washed in physiological saline. The rat skin stratum corneum was fixed upward in a transdermal diffusion cell (effective diffusion area 3.14 cm 2 ), 8 mL of PBS (pH 7.4) was added to the receiving cell as the receiving solution, 1 mL of each group of ellagic acid (Example 4) and ellagic acid nanoparticle solution (Example 3) was added to the supply chamber, and the mixture was stirred at 37°C and 400 r·min -1 The skin was then magnetically stirred at a speed of 1000 nm and protected from light for 12 hours. After the treatment, the skin was removed and washed with warm water to remove the residual preparation on the surface. The skin at the effective administration site was cut and fixed in a cryoembedding medium. Frozen sections were prepared and the drug distribution in the skin was observed under a fluorescence microscope.

[0048] The results of fluorescence microscopy of the skin after administration of ellagic acid and ellagic acid nanoparticles are shown in Figure 3 , it can be seen that 12 hours after administration, from the distribution in the skin, the fluorescence of the skin treated with ellagic acid is mainly distributed in the outermost stratum corneum and hair follicles, while the fluorescence of the skin treated with ellagic acid nanoparticles is not only distributed in the stratum corneum and hair follicles, but also observed to be distributed in the deep fascia layer of the skin. It can be seen that after ellagic acid is prepared as nanoparticles, it can be delivered deep into the skin, as deep as the fascia layer. In summary, ellagic acid nanoparticles have a strong affinity with the skin, reduce the barrier function of the skin, and can penetrate deep into the skin, reaching and targeting the deepest fascia layer of the skin, which may be an important reason why ellagic acid nanoparticles have excellent efficacy in preventing / treating hair loss after external use.

[0049] Example 9 Preparation of Ellagic Acid Nanoparticles for External Use

[0050] The obtained ellagic acid nanoparticles are prepared by the method of any one of Examples 1 or 3 or Claims 1 to 6, and used as raw materials in the preparation of external preparations, wherein the external preparations have the functions of preventing or treating medicines, cosmetics, and daily chemicals including the following effects: whitening, freckle removal, anti-oxidation, anti-aging, free radical scavenging, wrinkle removal, sun protection, enhancing skin elasticity, reducing pigmentation, moisturizing, antibacterial, antiviral, treating hair loss, treating white hair, removing skin grease, treating melanoma, and scar repairing, and the deep penetration of the drug is conducive to the exertion of the efficacy of these preparations. The external preparations include tinctures, gels, ointments, creams, patches, plasters, foams, lotions, films, films, etc., preferably foams.

Claims

1. Application of ellagic acid nanoparticles in the preparation of an external preparation with non-invasive deep skin drug delivery function, characterized in that: The ellagic acid nanoparticles are prepared by the following method: ellagic acid powder is added to a preparation solvent and prepared by ultrasonic process; the preparation solvent is pure water or an organic solvent or a mixed solution of an organic solvent and water, and the organic solvent is selected from one or more of ethanol, propylene glycol, butylene glycol, pentanediol, and glycerol; the ultrasonic process conditions are: ellagic acid feed concentration ≥1.0 mg / mL, ultrasonic power ≥100w, ultrasonic frequency ≥20kHz, ultrasonic temperature ≥30°C, and ultrasonic time ≥5min; after ultrasonication, the suspension is subjected to physical method to remove the solid part, and the physical method includes centrifugation, filtration, and sedimentation, and the supernatant is taken to obtain the ellagic acid nanoparticles.

2. The use according to claim 1, characterized in that: In the ultrasonic process conditions, the ultrasonic temperature is ≥35°C.

3. The use according to claim 2, characterized in that: The ultrasonic temperature in the ultrasonic process condition is 45°C.

4. The use according to claim 1, characterized in that: In the ultrasonic process conditions, the ellagic acid feed concentration is ≥2.5 mg / mL.

5. The use according to claim 4, characterized in that: The concentration of ellagic acid in the ultrasonic process is 20 mg / mL.

6. The use according to claim 1, characterized in that: The preparation solvent is water, or a mixed solution of an organic solvent and water, wherein the volume ratio of the organic solvent to water is 0:1-3:

1.

7. The use according to any one of claims 1 to 6, characterized in that: The external preparations are medicines, cosmetics, and daily chemical products.

8. The use according to claim 7, characterized in that: The external preparation is a tincture, a gel, an ointment, a cream, a patch, a plaster, a foam, a lotion, a film, and a patch.

9. The use according to claim 8, characterized in that: The external preparation is a foam, and its preparation method is as follows: prepare the ellagic acid nanoparticles in any one of claims 1 to 8 into a dispersion using an ultrasonic disperser; weigh a surfactant and add it to the dispersion, and simultaneously add a moisturizer and a stabilizer, and mix them completely and evenly; drip triethanolamine to neutralize the system until the pH value reaches 6.5-7.0, so as to form a stable drug-containing liquid foam matrix; put the prepared drug-containing liquid foam matrix into a foam pump bottle, disperse it through a foam pump to form a stable foam, and obtain the ellagic acid nanoparticle foam.

10. The use according to any one of claims 1 to 9, characterized in that: The topical preparation has one or more of the following functions: whitening, freckle removal, anti-oxidation, anti-aging, free radical scavenging, wrinkle removal, sun protection, enhancing skin elasticity, reducing pigmentation, moisturizing, antibacterial, antiviral, treating hair loss, treating gray hair, removing skin grease, treating melanoma, scar repair, and wound healing.

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