A silymarin nanocrystal-lipid complex nanoparticle, a preparation method and application thereof
Patent Information
- Application Number
- CN202610952010.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-06-29
- Publication Date
- 2026-08-21
AI Technical Summary
[0005]本发明的目的在于提供一种水飞蓟素纳米晶-脂质复合纳米粒及其制备方法和应用，用以解决现有水飞蓟素在化妆品及经皮给药领域中溶解性差、皮肤渗透性低、制剂稳定性不佳以及现有纳米载体载药量低、透皮增效有限的技术问题
本发明公开了一种水飞蓟素纳米晶-脂质复合纳米粒的制备方法，该方法区别于传统脂质体将药物包封于脂质双分子层或内水腔的负载模式，而是采用先制备水飞蓟素纳米晶内核、后进行磷脂包衣的核壳结构设计。本发明的技术效果主要来源于纳米晶化和磷脂界面包覆的协同作用：一方面，纳米晶技术将水飞蓟素粒径减小至100~400 nm，显著增大比表面积，提高水飞蓟素的饱和溶解度和溶出速率；另一方面，外层磷脂材料具有与皮肤角质层脂质相似的结构，可融合并扰乱角质层的有序排列，暂时增加脂质流动性，为纳米晶打开透皮通道。本发明改善了水飞蓟素水溶性差、稳定性不足和皮肤递送效率有限的问题，同时克服了单一纳米晶皮肤滞留时间短以及单一脂质体载药量受限、易泄漏等不足。
Smart Images
Figure CN122604634A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biotechnology, specifically relating to a silymarin nanocrystal-lipid composite nanoparticle, its preparation method, and its application. Background Technology
[0002] Silymarin (SM) is a flavonoid lignan active ingredient extracted from milk thistle fruit. Recent studies have shown that it possesses multiple benefits, including excellent anti-photoaging properties, free radical scavenging, tyrosinase inhibition, skin barrier repair, and inflammation soothing, demonstrating high development value in cosmetics such as sunscreens, whitening products, anti-aging products, and sensitive skin care. However, SM exhibits extremely poor water and lipid solubility, classifying it as a typical poorly soluble active ingredient. This makes it difficult for it to effectively penetrate the stratum corneum and accumulate at the epidermal and dermal target sites, resulting in low transdermal bioavailability. Furthermore, SM is prone to crystallization and uneven dispersion in conventional cosmetic matrices, exhibiting poor long-term storage stability, severely limiting its direct addition and application in high-value-added products such as serums, lotions, and masks. Therefore, developing a silymarin cosmetic composite carrier that combines high drug loading capacity, high skin permeability, and good stability has become a critical technological bottleneck that urgently needs to be overcome in this field.
[0003] To improve the transdermal bioavailability of styrax (SM), existing technologies mainly employ two strategies: one is to prepare it as nanocrystals, thereby increasing the dissolution rate by reducing particle size and increasing specific surface area; the other is to encapsulate it in liposomes or phospholipid complexes, utilizing the biomembrane affinity of phospholipids to promote transdermal absorption. However, pure nanocrystal formulations have drawbacks such as poor stability, short skin retention time, and potential irritation; while traditional liposomes, although exhibiting good biocompatibility, have limited encapsulation capacity for poorly soluble drugs like SM and are prone to drug leakage, making it difficult to simultaneously achieve the dual advantages of high drug loading and high permeability.
[0004] Currently, although a few studies have attempted to combine nanotechnology with phospholipid carriers, most methods involve directly preparing drug-loaded liposomes by dissolving the drug in an organic phase. The resulting system still uses liposomes as the primary component, with the drug mainly dispersed within the lipid membrane, thus failing to fundamentally address the issues of low drug loading and poor stability. Summary of the Invention
[0005] The purpose of this invention is to provide a silymarin nanocrystal-lipid composite nanoparticle, its preparation method, and its application, in order to solve the technical problems of poor solubility, low skin permeability, and poor formulation stability of silymarin in cosmetics and transdermal drug delivery, as well as the low drug loading capacity and limited transdermal synergistic effect of existing nanocarriers.
[0006] To achieve the above objectives, the present invention employs the following technical solution: This invention discloses a method for preparing silymarin nanocrystal-lipid composite nanoparticles, comprising the following steps: S1: Silymarin, stabilizer and water are mixed and then ground and freeze-dried in sequence to obtain silymarin nanocrystal powder; S2: Dissolve the phospholipid material in a solvent to obtain a phospholipid coating solution; S3: After removing the solvent by rotary evaporation of the phospholipid coating solution, a uniform phospholipid film is formed on the container wall used for rotary evaporation. Then, silymarin nanocrystal suspension is added for hydration to obtain silymarin nanocrystal-lipid composite nanoparticles. The silymarin nanocrystal suspension is obtained by mixing water and silymarin nanocrystal powder.
[0007] Further, in S1, the stabilizer is selected from one or more of sodium alginate, whey protein isolate, and Artemisia annua gum; The ratio of silymarin, stabilizer, and water is 1g:1g:20~60mL.
[0008] Further, in S1, the grinding process is carried out in a planetary ball mill; the grinding speed is 200~500 rpm, the grinding time is 2~6 h, the grinding media is zirconia beads with a particle size of 0.1~0.8 mm, and the mass ratio of grinding media to material is 200:1~100:1. The mass concentration of the silymarin nanocrystal suspension is 0.1%~2.0%.
[0009] Further, in S2, the phospholipid material is selected from one or more of soybean lecithin, cholesterol, egg yolk lecithin, and distearate phosphatidylcholine; The solvent is an organic solvent or an aqueous alcohol system; the organic solvent is selected from one or more of ethanol, methanol and chloroform.
[0010] Furthermore, in S2, the mass concentration of the phospholipid coating solution is 0.3% to 6.0%.
[0011] Furthermore, in S3, the mass ratio of the phospholipid material in the phospholipid coating solution to the silymarin nanocrystal powder in the silymarin nanocrystal suspension is 1:5 to 5:1.
[0012] Furthermore, in S3, the hydration temperature is 30~50℃ and the hydration time is 30~60 min.
[0013] The present invention also discloses a silymarin nanocrystal-lipid composite nanoparticle, characterized in that it is prepared by the above preparation method.
[0014] This invention also discloses the application of silymarin nanocrystal-lipid composite nanoparticles in the preparation of cosmetics or transdermal drug delivery formulations.
[0015] Furthermore, the cosmetic includes at least one of serum, lotion, and cream; the transdermal drug delivery formulation includes at least one of patch and ointment.
[0016] Compared with the prior art, the present invention has the following beneficial effects: This invention discloses a method for preparing silymarin nanocrystal-lipid composite nanoparticles. This method differs from the traditional liposome loading mode, which encapsulates the drug within a lipid bilayer or an inner water cavity. Instead, it employs a core-shell structure design, first preparing the silymarin nanocrystal core and then coating it with phospholipids. The technical effects of this invention mainly stem from the synergistic effect of nanocrystalization and phospholipid interface coating: on the one hand, nanocrystal technology reduces the silymarin particle size to 100-400 nm, significantly increasing the specific surface area and improving the saturated solubility and dissolution rate of silymarin; on the other hand, the outer phospholipid material has a structure similar to the lipids of the stratum corneum, which can fuse with and disrupt the orderly arrangement of the stratum corneum, temporarily increasing lipid fluidity and opening transdermal channels for the nanocrystals. This invention improves upon the problems of poor water solubility, insufficient stability, and limited skin delivery efficiency of silymarin, while overcoming the shortcomings of single nanocrystals (short skin retention time) and single liposomes (limited drug loading and easy leakage).
[0017] Furthermore, the method of this invention employs planetary grinding to prepare nanocrystals. The grinding process is conducted under mild conditions (200-500 rpm, room temperature operation), avoiding damage to the active components of silymarin by high temperatures or strong shear forces. Phospholipid coating can be achieved through self-assembly techniques (such as thin-film hydration or ethanol injection), which are mild, do not require vigorous emulsification, and have no significant impact on the crystal form and activity of the nanocrystals. The entire process has clearly defined and precisely controlled parameters, requires simple equipment, and is easy to scale up from the laboratory to industrial production. Attached Figure Description
[0018] Figure 1 Comparison of the appearance of silymarin nanocrystalline powder; Figure 2 These are comparative images of the appearance of the silymarin nanocrystal suspension of the present invention; Figure 3 The images show the appearance of the silymarin liposome suspension in Comparative Example 1 and the silymarin nanocrystal-lipid composite nanoparticle solutions prepared in Examples 1-3 of this invention. Figure 4 The figure shows the encapsulation efficiency and loading rate of the silymarin nanocrystal-lipid composite nanoparticles prepared in Experimental Example 4 of this invention. Where a - encapsulation ratio; b - load factor; Figure 5 The figure shows the cytotoxicity results of the silymarin nanocrystal-lipid composite nanoparticles prepared in Experimental Example 5 of this invention. Figure 6 This is a diagram showing the cellular uptake results of the silymarin nanocrystal-lipid composite nanoparticles prepared in Experimental Example 6 of this invention. Figure 7 The images show the skin permeability curve and the skin permeability test results at 36 h for the silymarin nanocrystal-lipid composite nanoparticles prepared in Experimental Example 7 of this invention. Wherein, a - skin permeability curve; b - skin permeability at 36 hours. Detailed Implementation
[0019] To enable those skilled in the art to understand the features and effects of the present invention, the terms and expressions used in the specification and claims are explained and defined in general below. Unless otherwise specified, all technical and scientific terms used herein have the ordinary meaning understood by those skilled in the art regarding the present invention, and in case of conflict, the definitions in this specification shall prevail.
[0020] The theories or mechanisms described and disclosed herein, whether right or wrong, should not in any way limit the scope of the invention, that is, the contents of the invention can be implemented without being limited by any particular theory or mechanism.
[0021] In this document, all features defined by numerical ranges or percentage ranges, such as numerical values, quantities, contents, and concentrations, are for the sake of brevity and convenience only. Accordingly, descriptions of numerical ranges or percentage ranges should be considered as covering and specifically disclosing all possible sub-ranges and individual numerical values (including integers and fractions) within those ranges.
[0022] In this article, unless otherwise specified, “contains,” “includes,” “containing,” “has,” or similar terms cover the meanings of “composed of” and “mainly composed of,” for example, “A contains a” covers the meanings of “A contains a and others” and “A contains only a.”
[0023] For the sake of brevity, not all possible combinations of the technical features in each implementation scheme or embodiment are described herein. Therefore, as long as there is no contradiction in the combination of these technical features, the technical features in each implementation scheme or embodiment can be combined arbitrarily, and all possible combinations should be considered within the scope of this specification.
[0024] S1: Silymarin, stabilizer and water are mixed and ground in a planetary ball mill. After grinding, the grinding media are separated, the filtrate is collected and freeze-dried to obtain silymarin nanocrystal powder. S2: Dissolve the phospholipid material in an organic solvent or an aqueous alcohol system, stir until completely dissolved, and prepare a phospholipid coating solution; S3: The phospholipid coating solution prepared in S2 is rotary evaporated to remove the organic solvent, so that a uniform phospholipid film is formed on the container wall. Then, silymarin nanocrystal suspension is added for hydration to obtain silymarin nanocrystal-lipid composite nanoparticles.
[0025] The silymarin nanocrystal suspension is obtained by mixing water and silymarin nanocrystal powder, with a mass concentration of 0.1% to 2.0% (w / v).
[0026] Preferably, in S1, the stabilizer is selected from one or more of sodium alginate, whey protein isolate, and Artemisia annua gum; the ratio of silymarin, stabilizer, and water is 1 g:0.1 g:20~60 mL.
[0027] Preferably, in S1, the grinding speed of the planetary ball mill is 200~500 rpm, the grinding time is 2~6 h, the grinding media is zirconia beads with a particle size of 0.1~0.8 mm, and the mass ratio of grinding media to material is 200:1~100:1.
[0028] Preferably, in S2, the phospholipid material is selected from one or more of soybean lecithin, cholesterol, egg yolk lecithin, and distearate phosphatidylcholine; the organic solvent is selected from one or more of ethanol, methanol, and chloroform.
[0029] Preferably, in S2, the mass concentration of the phospholipid coating solution is 0.3% to 6.0%.
[0030] Preferably, in S3, the mass ratio of the phospholipid material in the phospholipid coating solution to the silymarin nanocrystals in the silymarin nanocrystal suspension is 1:5 to 5:1.
[0031] Preferably, in S3, the hydration temperature is 30~50℃ and the hydration time is 30~60 min.
[0032] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.
[0033] The following examples use instruments and equipment conventional in the art. Experimental methods in the following examples, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. All raw materials used in the following examples are conventional commercially available products with specifications conventional in the art. In this specification and the following examples, unless otherwise specified, "%" refers to weight percentage, "parts" refers to parts by weight, and "ratio" refers to weight proportion.
[0034] Example 1 A method for preparing silymarin nanocrystal-lipid composite nanoparticles includes the following steps: S1: Weigh 0.1 g of sodium alginate and dissolve it in 20 mL of deionized water. Stir until completely dissolved to prepare a sodium alginate solution. Weigh 1.0 g of silymarin raw material and add it to the above solution (solid-liquid ratio 1:20, g / mL). Stir until homogeneous to obtain a premix. Transfer the premix to a 250 mL grinding jar, add 200 g of zirconia grinding beads with a particle size of 0.33 mm, place it in a planetary ball mill, and grind at 300 rpm for 2 h. After grinding, separate the zirconia beads with a 200-mesh sieve, collect the filtrate, and freeze-dry it to obtain silymarin nanocrystalline powder. S2: Weigh 1.0 g of cholesterol and 2.0 g of soybean lecithin according to the mass ratio of cholesterol to soybean lecithin = 1:2, dissolve them in an appropriate amount of chloroform, stir magnetically until completely dissolved, and prepare a phospholipid solution with a total lipid mass concentration of 3%. S3: Transfer the phospholipid solution prepared in S2 to a 50 mL round-bottom flask and remove chloroform by rotary evaporation in a 40℃ water bath, so that a uniform phospholipid film forms on the flask wall. Dissolve the silymarin nanocrystal powder from S1 in an appropriate amount of pure water to prepare a suspension (mass concentration of 1% (w / v)) according to a mass ratio of silymarin nanocrystals:cholesterol:soy lecithin = 1:1:2. Add this suspension to the above round-bottom flask and hydrate by rotary hydration in a 45℃ water bath for 30 min, allowing the phospholipid film to detach and self-assemble onto the surface of the nanocrystals, thus obtaining silymarin nanocrystal-lipid composite nanoparticles.
[0035] Example 2 A method for preparing silymarin nanocrystal-lipid composite nanoparticles includes the following steps: S1: Weigh 0.1 g of sodium alginate and dissolve it in 40 mL of deionized water. Stir until completely dissolved to prepare a sodium alginate solution. Weigh 1.0 g of silymarin raw material and add it to the above solution (solid-liquid ratio 1:40, g / mL). Stir until homogeneous to obtain a premix. Transfer the premix to a 250 mL grinding jar, add 200 g of zirconia grinding beads with a particle size of 0.33 mm, place it in a planetary ball mill, and grind at 300 rpm for 2 h. After grinding, separate the zirconia beads with a 200-mesh sieve, collect the filtrate, and freeze-dry it to obtain silymarin nanocrystalline powder. S2: Weigh 1.0 g of cholesterol and 2.0 g of soybean lecithin according to the mass ratio of cholesterol to soybean lecithin = 1:2, dissolve them in an appropriate amount of chloroform, stir magnetically until completely dissolved, and prepare a phospholipid solution with a total lipid mass concentration of 3%. S3: Transfer the phospholipid solution prepared in S2 to a 50 mL round-bottom flask and remove chloroform by rotary evaporation in a 40℃ water bath to form a uniform phospholipid film on the flask wall. Dissolve the silymarin nanocrystal powder from S1 in an appropriate amount of pure water at a mass ratio of 1% (w / v) to 1% (w / v), and add this suspension to the round-bottom flask. Hydrate by rotary evaporation in a 45℃ water bath for 30 min to allow the phospholipid film to detach and self-assemble onto the surface of the nanocrystals, thus obtaining silymarin nanocrystal-lipid composite nanoparticles.
[0036] Example 3 A method for preparing silymarin nanocrystal-lipid composite nanoparticles includes the following steps: S1: Weigh 0.1 g of sodium alginate and dissolve it in 60 mL of deionized water. Stir until completely dissolved to prepare a sodium alginate solution. Weigh 1.0 g of silymarin raw material and add it to the above solution (solid-liquid ratio 1:60, g / mL). Stir until homogeneous to obtain a premix. Transfer the premix to a 250 mL grinding jar, add 200 g of zirconia grinding beads with a particle size of 0.33 mm, place it in a planetary ball mill, and grind at 300 rpm for 2 h. After grinding, separate the zirconia beads with a 200-mesh sieve, collect the filtrate, and freeze-dry it to obtain silymarin nanocrystalline powder. S2: Weigh 1.0 g of cholesterol and 2.0 g of soybean lecithin according to the mass ratio of cholesterol to soybean lecithin = 1:2, dissolve them in an appropriate amount of chloroform, stir magnetically until completely dissolved, and prepare a phospholipid solution with a total lipid mass concentration of 3%. S3: Transfer the phospholipid solution prepared in S2 to a 50 mL round-bottom flask and remove chloroform by rotary evaporation in a 40℃ water bath to form a uniform phospholipid film on the flask wall. Dissolve the silymarin nanocrystal powder from S1 in an appropriate amount of pure water at a mass ratio of 1% (w / v) to 1% (w / v), and add this suspension to the round-bottom flask. Hydrate by rotary evaporation in a 45℃ water bath for 30 min to allow the phospholipid film to detach and self-assemble onto the surface of the nanocrystals, thus obtaining silymarin nanocrystal-lipid composite nanoparticles.
[0037] Example 4 A method for preparing silymarin nanocrystal-lipid composite nanoparticles includes the following steps: S1: Weigh 0.1 g of whey protein isolate, dissolve it in 20 mL of deionized water, and stir until completely dissolved to prepare a whey protein isolate solution; weigh 1.0 g of silymarin raw material and add it to the above solution (solid-to-liquid ratio 1:20, g / mL), stir well to obtain a premix. Transfer the premix to a 250 mL grinding jar, add 200 g of zirconia grinding beads with a particle size of 0.33 mm, place it in a planetary ball mill, and grind at 300 rpm for 2 h. After grinding, separate the zirconia beads with a 200-mesh sieve, collect the filtrate, and freeze-dry it to obtain silymarin nanocrystal powder; S2: Weigh 1.0 g of cholesterol and 2.0 g of soybean lecithin according to the mass ratio of cholesterol to soybean lecithin = 1:2, dissolve them in an appropriate amount of chloroform, stir magnetically until completely dissolved, and prepare a phospholipid solution with a total lipid mass concentration of 3%. S3: Transfer the phospholipid solution prepared in S2 to a 50 mL round-bottom flask and remove chloroform by rotary evaporation in a 40℃ water bath to form a uniform phospholipid film on the flask wall. Dissolve the silymarin nanocrystal powder from S1 in an appropriate amount of pure water at a mass ratio of 1% (w / v) to 1% (w / v), and add this suspension to the round-bottom flask. Hydrate by rotary evaporation in a 45℃ water bath for 30 min to allow the phospholipid film to detach and self-assemble onto the surface of the nanocrystals, thus obtaining silymarin nanocrystal-lipid composite nanoparticles.
[0038] Example 5 A method for preparing silymarin nanocrystal-lipid composite nanoparticles includes the following steps: S1: Weigh 0.1 g of whey protein isolate, dissolve it in 40 mL of deionized water, and stir until completely dissolved to prepare a whey protein isolate solution; weigh 1.0 g of silymarin raw material and add it to the above solution (solid-to-liquid ratio 1:40, g / mL), stir well to obtain a premix. Transfer the premix to a 250 mL grinding jar, add 200 g of zirconia grinding beads with a particle size of 0.33 mm, place it in a planetary ball mill, and grind at 300 rpm for 2 h. After grinding, separate the zirconia beads with a 200-mesh sieve, collect the filtrate, and freeze-dry it to obtain silymarin nanocrystal powder; S2: Weigh 1.0 g of cholesterol and 2.0 g of soybean lecithin according to the mass ratio of cholesterol to soybean lecithin = 1:2, dissolve them in an appropriate amount of chloroform, stir magnetically until completely dissolved, and prepare a phospholipid solution with a total lipid mass concentration of 3%. S3: Transfer the phospholipid solution prepared in S2 to a 50 mL round-bottom flask and remove chloroform by rotary evaporation in a 40℃ water bath to form a uniform phospholipid film on the flask wall. Dissolve the silymarin nanocrystal powder from S1 in an appropriate amount of pure water at a mass ratio of 1% (w / v) to 1% (w / v), and add this suspension to the round-bottom flask. Hydrate by rotary evaporation in a 45℃ water bath for 30 min to allow the phospholipid film to detach and self-assemble onto the surface of the nanocrystals, thus obtaining silymarin nanocrystal-lipid composite nanoparticles.
[0039] Example 6 A method for preparing silymarin nanocrystal-lipid composite nanoparticles includes the following steps: S1: Weigh 0.1 g of whey protein isolate, dissolve it in 60 mL of deionized water, and stir until completely dissolved to prepare a whey protein isolate solution; weigh 1.0 g of silymarin raw material and add it to the above solution (solid-liquid ratio 1:60, g / mL), stir well to obtain a premix. Transfer the premix to a 250 mL grinding jar, add 200 g of zirconia grinding beads with a particle size of 0.33 mm, place it in a planetary ball mill, and grind at 300 rpm for 2 h. After grinding, separate the zirconia beads with a 200-mesh sieve, collect the filtrate, and freeze-dry it to obtain silymarin nanocrystal powder; S2: Weigh 1.0 g of cholesterol and 2.0 g of soybean lecithin according to the mass ratio of cholesterol to soybean lecithin = 1:2, dissolve them in an appropriate amount of chloroform, stir magnetically until completely dissolved, and prepare a phospholipid solution with a total lipid mass concentration of 3%. S3: Transfer the phospholipid solution prepared in S2 to a 50 mL round-bottom flask and remove chloroform by rotary evaporation in a 40℃ water bath to form a uniform phospholipid film on the flask wall. Dissolve the silymarin nanocrystal powder from S1 in an appropriate amount of pure water at a mass ratio of 1% (w / v) to 1% (w / v), and add this suspension to the round-bottom flask. Hydrate by rotary evaporation in a 45℃ water bath for 30 min to allow the phospholipid film to detach and self-assemble onto the surface of the nanocrystals, thus obtaining silymarin nanocrystal-lipid composite nanoparticles.
[0040] Comparative Example 1 Weigh 2.0 g of soybean lecithin and 1.0 g of cholesterol, dissolve them in chloroform, and evaporate them in a water bath at 40°C to form a phospholipid film. Add silymarin raw material solution (simulating the proportion of nanocrystals), hydrate at 45°C, and obtain a silymarin liposome suspension.
[0041] Experimental Example 1 The appearance of the silymarin nanocrystal powder and the water dispersibility of the silymarin nanocrystals in Examples 1-6 were photographed.
[0042] The results are as follows Figure 1 As shown, compared to the raw silymarin, the silymarin nanocrystal powder has a lighter color, possibly because the stabilizers (sodium alginate and whey protein isolate) are themselves white / off-white, and after encapsulation, they physically encapsulate the raw material. The raw silymarin exhibits severe agglomeration, forming irregular lumps with poor flowability. In contrast, the silymarin nanocrystal powder stabilized by sodium alginate and whey protein isolate is a uniform, loose powder, with significantly less agglomeration than the raw material. The silymarin nanocrystals prepared in this invention can greatly improve the water dispersibility of silymarin, such as... Figure 2 As shown in Table 1, silymarin raw material powder was completely insoluble, with a large amount of orange-yellow particles precipitating out, forming obvious solid-liquid separation. The sodium alginate-stabilized silymarin nanocrystal solution was uniform and free of precipitation, exhibiting better water dispersibility than whey protein isolate.
[0043] Table 1 Appearance characteristics of silymarin nanocrystal suspension
[0044] Experiment Example 2 The silymarin nanocrystals and their silymarin nanocrystal-lipid composite nanoparticles from Examples 1-6 were diluted to appropriate concentrations, and the average particle size and polydispersity index (PDI) were determined using a dynamic light scattering particle size analyzer.
[0045] The average particle size and polydispersity index of silymarin nanocrystals and their silymarin nanocrystal-lipid composite nanoparticles are shown in Table 2. Sodium alginate, as a stabilizer, can prepare silymarin nanocrystals with smaller particle size and more uniform distribution. After lipid coating, the particle size is still maintained at 230-260 nm. The silymarin nanocrystals stabilized by whey protein isolate have a larger particle size. After lipid coating, the particle size of the whey protein isolate-stabilized silymarin nanocrystal-lipid composite nanoparticles exceeds 450 nm.
[0046] Table 2. Average particle size and PDI of silymarin nanocrystals and their silymarin nanocrystal-lipid composite nanoparticles
[0047] Experimental Example 3 Take 4 mL each of the silymarin nanocrystal-lipid composite nanoparticles prepared in Examples 1-3 and the dispersion of Comparative Example 1, and add them to transparent glass sample bottles of the same specifications. After sealing, let them stand at room temperature. After standing, observe the appearance of each sample with the naked eye, including color, uniformity, precipitation, flocculation and stratification, and take pictures for recording.
[0048] The results are as follows Figure 3 As shown, all samples exhibited a pale yellow, milky dispersion. The comparative SM-LiP sample showed a noticeable sediment layer at the bottom, indicating relatively poor dispersion stability. In contrast, the silymarin nanocrystal-lipid composite nanoparticle sample stabilized by sodium alginate showed a more uniform overall dispersion, with no obvious large-scale precipitation or phase separation, suggesting that the nanocrystal-lipid composite technology helps improve the dispersion stability of silymarin in the aqueous phase.
[0049] Experiment Example 4 Take 1 mL of the silymarin nanocrystal-lipid composite nanoparticle suspension from Examples 1-3, place it in an ultrafiltration centrifuge tube with a molecular weight cutoff of 30 kDa, centrifuge at 3,000 rpm for 30 min, and collect the filtrate as the free drug solution; take another equal volume of the suspension, add methanol to demulsify and make up to an appropriate volume, and measure the absorbance at a wavelength of 288 nm using ultraviolet spectrophotometry, calculate the total drug concentration according to the standard curve, and at the same time, take the above filtrate directly or after appropriate dilution and measure the free drug concentration at the same wavelength.
[0050] Encapsulation efficiency (EE%) = (Total drug content - Free drug content) / Total drug content × 100% Loading rate (DL%) = (Amount of encapsulated drug) / Total mass of nanocrystal-lipid composite nanoparticles × 100% The results are as follows Figure 4As shown, when the material-liquid ratio is 1:60, the encapsulation efficiency and loading rate of silymarin nanocrystal-lipid composite nanoparticles stabilized by sodium alginate are approximately 81% and 52%, respectively, which are the optimal values among the three ratios.
[0051] Experimental Example 5 The cytocompatibility of silymarin nanocrystals and silymarin nanocrystal-lipid composite nanoparticles from Example 3 was evaluated: Human skin fibroblasts (HDF) were cultured to the logarithmic growth phase and seeded into 96-well plates. Different concentrations (10, 20, 50 μg / mL) of silymarin nanocrystals, silymarin nanocrystal-lipid composite nanoparticles, and blank culture medium were added and treated for 24 h. Cell viability was determined using the CCK-8 assay.
[0052] The results are as follows Figure 5 The results showed that at concentrations of 10, 20, and 50 μg / mL, the cell viability of all experimental groups remained above 90%, with no significant difference from the control group. This indicates that both formulations have good biocompatibility and no obvious cytotoxicity within the experimental concentration range, and can be used for subsequent cell uptake studies.
[0053] Experimental Example 6 The cellular uptake capacity of silymarin nanocrystals and silymarin nanocrystal-lipid composite nanoparticles from Example 3 was evaluated: Human skin fibroblasts were seeded in 24-well plates, and coumarin 6-labeled silymarin nanocrystals and silymarin nanocrystal-lipid composite nanoparticles were added, respectively. After 12 h, the incubation was terminated, and the cells were collected by centrifugation at 1000 r / min for 5 min. 100 µL of cell lysis buffer was added, the cells were vortexed for 1 min, incubated at 4℃ for 40 min, 100 µL of PBS was added, and the cells were centrifuged at 13000 r / min for 20 min. The fluorescence intensity of the supernatant was measured.
[0054] The results are as follows Figure 6 As shown, lipid coating significantly improved the cellular uptake efficiency of nanocrystals: the fluorescence intensity of the SM-NC-LIP group was nearly twice that of the SM-NC group, indicating that the modification of the lipid layer promoted the uptake of the formulation by cells.
[0055] Experimental Example 7 The skin permeability of silymarin nanocrystals and silymarin nanocrystal-lipid composite nanoparticles from Comparative Example 1 and Example 3 was evaluated: using a Franz diffusion cell with ex vivo porcine skin as the transdermal barrier, the effective diffusion area was 0.2827 cm². Coumarin-6-labeled samples were added to the supply cell, and PBS containing 20% ethanol was used in the receiving cell. The mixture was stirred at 37°C. Samples were taken at different time points (1, 2, 4, 8, 12, 24, and 36 h), and the fluorescence intensity was measured using a microplate reader to calculate the skin permeability.
[0056] The results are as follows Figure 7 As shown, within 36 h, the cumulative skin permeability of the SM-NC-LIP group (approximately 10.5%) was significantly higher than that of the SM-NC group (approximately 8.5%) and the SM-LIP group (approximately 5%). SM-NC-LIP showed a continuous and rapid penetration trend throughout the experiment, indicating that the silymarin nanocrystal-lipid composite nanoparticles significantly improved the skin delivery efficiency of silymarin through the synergistic effect of the nanocrystal penetration effect and the lipid layer fusion effect, providing a key basis for the development of its skin drug delivery formulation.
[0057] The preparation method disclosed in this invention includes the following steps: using sodium alginate (SA) or whey protein isolate (WPI) as stabilizers, silymarin nanocrystals (SM-NC) are prepared, and further, silymarin nanocrystal-lipid composite nanoparticles (SM-NC-LIP) are constructed using lipid encapsulation technology. The optimized silymarin nanocrystal-lipid composite nanoparticles have a particle size of approximately 230-260 nm and a PDI < 20%, which not only significantly improves the water dispersibility and stability of silymarin but also increases the encapsulation efficiency and loading rate of silymarin. In vitro evaluation results show that these nanoparticles have no significant cytotoxicity and can significantly improve the uptake efficiency of human skin fibroblasts. Franz diffusion cell transdermal experiments confirmed that the cumulative skin permeability of silymarin nanocrystal-lipid composite nanoparticles is significantly superior to that of traditional silymarin liposomes, enabling efficient penetration of the stratum corneum for active ingredient delivery. This invention solves the key problems of poor water solubility and low transdermal bioavailability of silymarin, providing a novel silymarin delivery system that combines high drug loading, high stability, and biocompatibility. It can be used to prepare skincare products such as serums, lotions, and creams with antioxidant, anti-inflammatory, and soothing repair properties, and has broad market application prospects.
[0058] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.
Claims
1. A method for preparing silymarin nanocrystal-lipid composite nanoparticles, characterized in that, Includes the following steps: S1: Silymarin, stabilizer and water are mixed and then ground and freeze-dried in sequence to obtain silymarin nanocrystal powder; S2: Dissolve the phospholipid material in a solvent to obtain a phospholipid coating solution; S3: After removing the solvent by rotary evaporation of the phospholipid coating solution, a uniform phospholipid film is formed on the container wall used for rotary evaporation. Then, silymarin nanocrystal suspension is added for hydration to obtain silymarin nanocrystal-lipid composite nanoparticles. The silymarin nanocrystal suspension is obtained by mixing water and silymarin nanocrystal powder.
2. The method for preparing silymarin nanocrystal-lipid composite nanoparticles according to claim 1, characterized in that, In S1, the stabilizer is selected from one or more of sodium alginate, whey protein isolate, and Artemisia argyi gum; The ratio of silymarin, stabilizer, and water is 1g:1g:20~60mL.
3. The method for preparing silymarin nanocrystal-lipid composite nanoparticles according to claim 1, characterized in that, In S1, the grinding process is carried out in a planetary ball mill; the grinding speed is 200~500 rpm, the grinding time is 2~6 h, the grinding media is zirconia beads with a particle size of 0.1~0.8 mm, and the mass ratio of grinding media to material is 200:1~100:
1. The mass concentration of the silymarin nanocrystal suspension is 0.1%~2.0%.
4. The method for preparing silymarin nanocrystal-lipid composite nanoparticles according to claim 1, characterized in that, In S2, the phospholipid material is selected from one or more of soybean lecithin, cholesterol, egg yolk lecithin, and distearate phosphatidylcholine; The solvent is an organic solvent or an aqueous alcohol system; the organic solvent is selected from one or more of ethanol, methanol and chloroform.
5. The method for preparing silymarin nanocrystal-lipid composite nanoparticles according to claim 1, characterized in that, In S2, the mass concentration of the phospholipid coating solution is 0.3%~6.0%.
6. The method for preparing silymarin nanocrystal-lipid composite nanoparticles according to claim 1, characterized in that, In S3, the mass ratio of phospholipid material in the phospholipid coating solution to silymarin nanocrystal powder in the silymarin nanocrystal suspension is 1:5~5:
1.
7. The method for preparing silymarin nanocrystal-lipid composite nanoparticles according to claim 1, characterized in that, In S3, the hydration temperature is 30~50℃ and the hydration time is 30~60 min.
8. A silymarin nanocrystal-lipid composite nanoparticle, characterized in that, It is prepared by the preparation method described in any one of claims 1 to 7.
9. The application of the silymarin nanocrystal-lipid composite nanoparticles according to claim 8 in the preparation of cosmetics or transdermal drug delivery formulations.
10. The application of the silymarin nanocrystal-lipid composite nanoparticles according to claim 9 in the preparation of cosmetics or transdermal drug delivery formulations, characterized in that, The cosmetics include at least one of serums, lotions, and creams; the transdermal drug delivery formulations include at least one of patches and ointments.