L-muscle peptide-sophorolipid micelles, and a preparation method and application thereof

Abstract

This invention belongs to the field of skincare technology, and relates to an L-carnosine-sophorolipid micelle, its preparation method, and its application. The method involves dissolving sophorolipid in methanol, ethanol, or acetone, and dissolving L-carnosine in an aqueous solution with a pH of 2-7. The mixtures are stirred at a mass ratio of 3:1 to 8:1, and then vacuum rotary evaporation is performed at -0.06 to -0.08 MPa and 30-70°C to remove organic solvents, forming a thin film. The film is then hydrated with physiological saline, purified using a 1000 Da ultrafiltration membrane, and freeze-dried to obtain the product. The resulting micelles have uniform particle size and an encapsulation efficiency of up to 99.34%, which can improve the carnosinase resistance and transdermal absorption efficiency of L-carnosine, while synergistically retaining its free radical scavenging, anti-inflammatory, antibacterial, and long-lasting moisturizing effects. This invention features a simple and controllable process, solving the problems of poor stability and low bioavailability of L-carnosine, and is suitable for topical skincare products, possessing significant application value.

Description

Technical Field

[0001] This invention belongs to the field of skin care technology, and specifically relates to an L-carnosine-sophorolipid micelle, its preparation method and application. Background Technology

[0002] In the skincare industry, as consumers increasingly demand highly effective and safe skincare ingredients, bioactive ingredients have gained widespread attention due to their combination of gentleness and functionality. The stability, transdermal absorption efficiency, and bioavailability of active ingredients are core factors determining the efficacy of skincare products. Most single active ingredients or simple compound systems in current technologies cannot simultaneously meet these requirements, limiting their application in topical skincare products.

[0003] L-Carnosine is a natural dipeptide composed of β-alanine and histidine. As a novel, highly effective, and low-toxicity biological antioxidant, it possesses significant skincare value: it effectively scavenges reactive oxygen species such as superoxide anions and hydroxyl radicals, as well as peroxyl free radicals. It also exhibits multiple functions including anti-glycation, intracellular buffering, tissue repair, enhanced immune response, and anti-apoptosis, demonstrating outstanding performance in preventing skin aging and brightening skin tone. However, L-Carnosine has inherent technical limitations: firstly, as a small-molecule dipeptide, it is easily degraded by specific carnosinases in vivo, leading to insufficient stability when applied in vitro; secondly, L-Carnosine has high water solubility but weak ability to cross the skin's natural biomembrane barrier, resulting in low bioavailability in topical skincare products, severely affecting its full skincare efficacy. Therefore, it is urgent to improve its stability and transdermal performance through reasonable structural modification or carrier encapsulation technology.

[0004] Sophorolipids are glycolipid biosurfactants produced by yeast metabolism. Due to their excellent surface activity, mild and non-irritating properties, anti-inflammatory and antibacterial effects, and moisturizing properties, they are widely used in cosmetics as foaming agents, solubilizers, dispersants, detergents, and emulsion stabilizers. More importantly, sophorolipids possess a unique amphiphilic structure; when the concentration reaches the critical micelle concentration, they can self-assemble into micelles. Their internal structure can serve as a carrier for active ingredients, effectively improving the stability and bioavailability of active substances. This makes them particularly suitable for the development of new skincare formulations such as microcapsules, beads, and micelles, demonstrating significant advantages in the personal care field.

[0005] From the perspective of synergistic efficacy, the antioxidant and anti-glycation effects of L-carnosine and the moisturizing, anti-inflammatory, antibacterial, and carrier properties of sophorolipids are theoretically complementary. If the two can be rationally combined, it is hoped that they can protect L-carnosine from degradation and enhance its transdermal absorption capacity, while achieving synergistic skincare effects. However, current technologies have not reported on the preparation of functional skincare carriers using L-carnosine and sophorolipids in combination. There is a lack of technical solutions that can simultaneously address the problems of poor stability and low transdermal absorption efficiency of L-carnosine, thus failing to meet the market demand for highly effective synergistic skincare products.

[0006] In summary, the application of L-carnosine in existing technologies is limited by insufficient stability and poor transdermal absorption. Furthermore, the carrier potential of sophorolipids has not been combined with the skincare benefits of L-carnosine, leaving a technological gap in their combined use and hindering breakthroughs in the efficacy of related skincare products. Therefore, developing a technical solution that can effectively improve the stability and transdermal performance of L-carnosine while simultaneously leveraging their synergistic skincare effects has become a pressing technical problem to be solved in this field. Summary of the Invention

[0007] To address the problems in the background technology, this invention proposes an L-carnosine-sophorolipid micelle, its preparation method, and its application. Based on the self-assembly characteristics of sophorolipid, a thin-film hydration method is used to rapidly and easily prepare L-carnosine-sophorolipid micelle molecules with uniform particle size. This solves the problems of L-carnosine being easily degraded by specific carnosinases in vivo and having low bioavailability due to its difficulty in crossing the skin barrier during transdermal absorption. Simultaneously, based on the different skincare effects of L-carnosine and sophorolipid, a synergistic skincare effect through topical application is achieved.

[0008] The technical solution adopted by this invention to solve its technical problem is: to provide a method for preparing L-carnosine-sophorolipid micelles, comprising the following steps:

[0009] Sophorolipid was dissolved in an organic solvent to obtain a sophorolipid solution;

[0010] L-carnosine was dissolved in water to obtain an L-carnosine aqueous solution;

[0011] Mix the sophorolipid solution with the L-carnosine aqueous solution;

[0012] Remove organic solvents to form a thin film;

[0013] The membrane was hydrated with physiological saline to form a micelle solution;

[0014] The micelle solution was purified to remove unreacted L-carnosine, and the retentate was lyophilized to obtain L-carnosine-sophorolipid micelles.

[0015] Furthermore, the organic solvents include methanol, ethanol, and / or acetone.

[0016] Furthermore, the pH value of the L-carnosine aqueous solution is between 2 and 7.

[0017] Furthermore, the mass ratio of sophorolipid to L-carnosine is 3:1 to 8:1.

[0018] Furthermore, the step of removing the organic solvent is vacuum rotary evaporation at a vacuum level of -0.06 MPa to -0.08 MPa and a temperature of 30°C to 70°C.

[0019] Furthermore, the amount of physiological saline added is 1-1.5 times the volume; the hydration time is 8-12 minutes.

[0020] Furthermore, the purification step includes ultrafiltration using a 1000-1500 Da ultrafiltration membrane.

[0021] The present invention also provides an L-carnosine-sophorolipid micelle, which is obtained by the above preparation method.

[0022] Furthermore, the mass ratio of sophorolipid to L-carnosine is 3:1 to 8:1.

[0023] This invention also provides an application of L-carnosine-sophorolipid micelles in skincare products.

[0024] The beneficial effects of this invention are:

[0025] This invention leverages the self-assembly properties of sophorolipids to rapidly and easily prepare L-carnosine-sophorolipid micelles with uniform particle size using a thin-film hydration method. This addresses the issues of L-carnosine's easy degradation by specific carnosinases in vivo and its low bioavailability due to difficulty in crossing the skin barrier during transdermal absorption. Furthermore, based on the different skincare effects of L-carnosine and sophorolipids, a synergistic skincare effect can be achieved through topical application. Attached Figure Description

[0026] Figure 1 Appearance A and Tyndall effect B of L-carnosine-sophorolipid micelles;

[0027] Figure 2 This is a particle size distribution diagram of L-carnosine-sophorolipid micelles;

[0028] Figure 3 TEM image of L-carnosine-sophorolipid micelles;

[0029] Figure 4 This is a graph showing the results of the anti-inflammatory activity test. Detailed Implementation

[0030] The technical solutions of the embodiments of the invention will be clearly and completely described below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are merely for explaining the invention and not for limiting the invention. Furthermore, it should be noted that, for ease of description, only the parts related to the invention are shown in the accompanying drawings, not all of the structures.

[0031] Example 1:

[0032] Sophorolipid and L-carnosine were dissolved in methanol and aqueous solution (pH=5.0), respectively, with a mass ratio of 5:1. The sophorolipid solution was slowly added dropwise to the L-carnosine solution, and the mixture was stirred for 30 min. The mixture was transferred to a round-bottom rotary evaporator flask, and the evaporation temperature was set to 50 °C and the vacuum degree to -0.07 MPa to remove the methanol solution. A thin film was formed in the evaporator flask, and one volume of physiological saline was added. Hydration was continued for 10 min. After cooling to room temperature, the mixture was passed through a 1000 Da ultrafiltration membrane to remove unreacted carnosine. The retentate was lyophilized to obtain L-carnosine-sophoralipid micelles. The micelles were dissolved in water and their appearance was observed. Figure 1 The micelle solution was transparent and clear, with a distinct Tyndall effect. The encapsulation efficiency was measured to be 97.45%, particle size: 92.95 ± 1.42 nm, and PDI: 0.12 ± 0.012. Figure 2 This is a particle size distribution diagram. Figure 3 This is a TEM image. The micelles are spherical, uniform in size, and well-dispersed.

[0033] Example 2:

[0034] Sophorolipids and L-carnosine were dissolved in ethanol and aqueous solution (pH=2.0), respectively, with a mass ratio of 3:1. The sophorolipid solution was slowly added dropwise to the L-carnosine solution, and the mixture was stirred for 30 min. The mixture was transferred to a round-bottom rotary evaporator flask, and the evaporation temperature was set to 30 °C and the vacuum degree to -0.08 MPa. After removing the ethanol solution, a thin film was formed in the evaporator flask. One volume of physiological saline was added, and hydration was continued for 10 min. After cooling to room temperature, the mixture was passed through a 1000 Da ultrafiltration membrane to remove unreacted carnosine. The retentate was lyophilized to obtain L-carnosine-sophoralipin micelles. The encapsulation efficiency was 95.34%, the particle size was 91.77±1.05 nm, and the PDI was 0.12±0.011.

[0035] Example 3:

[0036] Sophorolipids and L-carnosine were dissolved in acetone and aqueous solution (pH=7.0), respectively, with a mass ratio of 8:1. The sophorolipid solution was slowly added dropwise to the L-carnosine solution, and the mixture was stirred for 30 min. The mixture was transferred to a round-bottom rotary evaporator flask, and the evaporation temperature was set to 70 ℃, with a vacuum of -0.05 MPa. After removing the acetone solution, a thin film was formed in the evaporator flask. One volume of physiological saline was added, and hydration was continued for 10 min. After cooling to room temperature, the mixture was passed through a 1000 Da ultrafiltration membrane to remove unreacted carnosine. The retentate was lyophilized to obtain L-carnosine-sophoralipin micelles. The encapsulation efficiency was 99.34%, the particle size was 97.77±3.01 nm, and the PDI was 0.13±0.017.

[0037] Comparative Example 1:

[0038] Dissolving sophorolipids in water and performing the same procedures as in Example 1 resulted in micelle encapsulation of only 35.1%. This may be because the sophorolipids used in the experiment were mixed-type, containing lactone-type sophorolipids, which are insoluble in water and cannot fully contact L-carnosine. Consequently, this portion of the sophorolipids could not participate in the reaction and thus did not form micelles. Acidic sophorolipids, on the other hand, are readily soluble in water and can fully react with L-carnosine to form micelles. The encapsulation rate is positively correlated with the ratio of the two types of sophorolipids. If the sophorolipids in Comparative Example 1 were dissolved in ethyl acetate, the result was similar to Comparative Example 1, with an encapsulation rate of only 35.5%, indicating that only acidic sophorolipids participated in the reaction. This demonstrates that the solvent used to dissolve sophorolipids plays a crucial role in micelle formation.

[0039] Comparative Example 2:

[0040] Sophorolipids and L-carnosine were dissolved in methanol and aqueous solution (pH=1.0), respectively, with a mass ratio of 5:1. The remaining procedures were the same as in Example 1. No micelles formed under these conditions. This may be because, in a strongly acidic system, the glycosidic bonds in the sophorolipid structure are easily hydrolyzed, producing monosaccharides or disaccharides and fatty chains, losing their amphiphilic properties and thus failing to self-assemble into micelles. If L-carnosine from Comparative Example 2 was dissolved in an aqueous solution (pH=9), with the remaining conditions the same as in Example 1, the resulting micelle encapsulation rate was 64.5%. This may be because, in alkaline solutions, the carboxyl groups in both L-carnosine and acidic sophorolipids undergo deprotonation and become negatively charged, resulting in a strong charge repulsion between them. This indicates that the pH of the aqueous solution used to dissolve L-carnosine has a significant impact on micelle formation.

[0041] Comparative Example 3:

[0042] Sophorolipid and L-carnosine were dissolved in methanol and aqueous solution (pH=5.0), respectively, with a mass ratio of 5:1. The sophorolipid solution was slowly added dropwise to the L-carnosine solution, and the mixture was stirred for 30 min. The mixture was transferred to a round-bottom rotary evaporator flask, and the rotary evaporation temperature was set to 80 °C, with a vacuum of -0.07 MPa. The remaining operations were the same as in Example 1. Micelles failed to form under these conditions. Two possible reasons are: 1. High temperature increases the entropy of the solvent, making molecules more inclined to disperse rather than aggregate; 2. Excessive temperature causes the methanol solvent to boil violently, which is not conducive to micelle formation. If the rotary evaporation temperature was set to 20 °C, the encapsulation efficiency was 98.1%, the particle size was 110.7±12.01 nm, and the PDI was 0.31±0.012. Compared to Example 1, the micelles formed at this time had larger particle sizes, but lower uniformity. This may be because the rotary evaporation temperature was too low, and the rotary evaporation time was increased, allowing sophorolipid molecules to continue to aggregate on the L-carnosine-sopholipid micelle molecules. Clearly, rotary evaporation temperature is a key factor in micelle formation.

[0043] Comparative Example 4:

[0044] Sophorolipids and L-carnosine were dissolved in methanol and aqueous solution (pH=5.0), respectively, with a mass ratio of 1:1. Other conditions were the same as in Example 1. The encapsulation efficiency was measured to be 32%. This is likely because sophorolipids encapsulate L-carnosine in the micelle structure. If the ratio is 1:1, the relative amount of sophorolipids is too small, and some L-carnosine cannot be encapsulated and thus cannot form micelles. If the mass ratio of sophorolipids to L-carnosine in Comparative Example 4 is set to 10:1, the encapsulation efficiency remains at 99.1%. Clearly, further increasing the amount of sophorolipids leads to a stable encapsulation efficiency, with carnosine almost completely encapsulated. Therefore, too little sophorolipids results in a low micelle encapsulation efficiency; too much sophorolipids lead to waste.

[0045] Performance testing:

[0046] Carnosinase-1 resistance test:

[0047] Table 1 Results of carnosinase 1 hydrolysis experiment

[0048]

[0049] Carnosin-1 solution was mixed and incubated with L-carnosine solution (control group 1), a physical mixture of L-carnosine and sophorolipid (control group 2), and the L-carnosine-sophoracil micelle solution prepared in Example 1 (experimental group 1) in a 30 °C water bath, keeping the total amount of L-carnosine consistent in the control and experimental groups. Samples were taken at 0 min, 10 min, and 60 min, 10% sulfosalicylic acid was added, and the mixture was incubated on ice for 30 min. After centrifugation, the supernatant was collected, and 1% trifluoroacetic acid, phthalaldehyde, and Tris buffer were added. The mixture was incubated for another 30 min, and the fluorescence value was detected using a multifunctional microplate reader. The laser light source was 360 nm, and the emission light source was 465 nm. The histidine content was calculated based on the fluorescence value, which reflects the degree of carnosine hydrolysis, as detailed in Table 1. In control group 1, L-carnosine was rapidly hydrolyzed by carnosin-1 within 10 min, reaching a hydrolysis degree of 61.3%, and was completely hydrolyzed within 60 min. In control group 2, the degree of hydrolysis of carnosine was similar to that in control group 1, indicating that the simple addition of sophorolipids had no significant protective effect on L-carnosine. In experimental group 1, the degree of hydrolysis was significantly reduced at 10 min and 60 min, indicating that micelle formation can significantly improve the stability of L-carnosine in the carnosinase system.

[0050] Transdermal test:

[0051] Table 2. Intradermal retention of L-carnosine

[0052]

[0053] Using a Franz diffusion cell, transdermal experiments were conducted on pig skin at 1 h, 2 h, 4 h, and 8 h, following a pig skin permeation model. Since L-carnosine has difficulty penetrating the skin barrier, it was used as a marker active ingredient, and its retention in the skin was determined using high-performance liquid chromatography (HPLC) to characterize the transdermal efficiency of the active ingredient. The experiment used an aqueous solution of L-carnosine as control group 1, a physical mixture of L-carnosine and sophorolipids as control group 2, and the L-carnosine-sophoracil micelles prepared in Example 1 as experimental group 1, ensuring a consistent total L-carnosine content in each group. Each group was measured in triplicate, and the average value was taken (see Table 2). Only a small amount of L-carnosine permeated the skin in control group 1. Although sophorolipids were added to control group 2, L-carnosine and sophorolipids are immiscible, and only a small amount of L-carnosine still permeated the skin. The significantly increased L-carnosine content in experimental group 1 demonstrated good micelle permeability, contributing to the bioavailability of the active ingredient when applied topically.

[0054] Free radical scavenging experiment:

[0055] Table 3 DPPH free radical scavenging rate

[0056]

[0057] L-carnosine (experimental group 1) and L-carnosine-sophorolipid micelles (experimental group 2) were added to an ethanol solution containing DPPH (the absolute amount of L-carnosine in both groups was consistent, and the carnosine concentration in both groups was 0.1 mol / L). The mixture was thoroughly mixed and reacted at room temperature in the dark for 30 min, after which the absorbance was read at 517 nm. DPPH + buffer was used as a control group. The free radical scavenging rate (%) was calculated as [1 - A517 of experimental group / A517 of control group] × 100% (Table 3). The scavenging rate of experimental group 1 was 54.2%, and that of experimental group 2 was 49.3%, slightly lower than that of experimental group 1. This may be because L-carnosine is located inside the micelles, and sophorolipids block the scavenging rate of L-carnosine. However, the difference between the two groups is not significant, and both still retain good free radical scavenging capabilities.

[0058] Anti-inflammatory experiment: Interleukin-1α was released from keratinocytes and used to treat an epithelial reconstruction model. Cells treated only with UV light served as the control group. Cells treated with L-carnosine and L-carnosine-sophorolipid prepared in Example 1 (keeping the absolute amount of L-carnosine consistent in both groups) served as experimental groups 1 and 2, respectively. The interleukin-1α levels in the three groups were tested, and the results are shown in […]. Figure 4 The results showed that both L-carnosine and L-carnosine-sophorolipid micelles could inhibit the production and release of interleukin-1α by UV-stimulated keratinocytes, exhibiting good anti-inflammatory activity. Meanwhile, the anti-inflammatory activity of L-carnosine-sophorolipid micelles was slightly higher than that of pure L-carnosine, possibly due to the addition of sophorolipids, which provided a certain degree of synergistic anti-inflammatory activity.

[0059] Antibacterial test:

[0060] Table 4 Results of Staphylococcus aureus antibacterial test

[0061]

[0062] Using a sterile cotton swab, apply a Staphylococcus aureus suspension at a concentration of 5×10⁵ CFU / mL to 5×10⁶ CFU / mL onto an agarose agar plate and let it air dry for 5 minutes at room temperature. Take sterile filter paper and add L-carnosine (control group 1), sophorolipid (control group 2), and L-carnosine-sophorolipid micelles prepared in Example 1 (experimental group 1) to the filter paper, respectively. The mass of sophorolipid in control group 2 and experimental group 1 should be consistent. Place the filter paper in a sterile Petri dish and dry it in an oven for later use. Place four filter paper discs containing the test sample onto the surface of the agar plate, with a center-to-center distance of at least 25 mm and a distance of at least 15 mm from the periphery of the plate. Cover the Petri dish and incubate at 37 ℃ for 18 h. Measure the diameter of the inhibition zone with calipers. Perform three replicates and take the average value. See Table 4 for details. L-carnosine exhibited a weak antibacterial effect. The diameters of the inhibition zones in control group 2 and experimental group 1 were similar, indicating that L-carnosine-sophorolipid still retained good antibacterial properties.

[0063] Moisturizing experiment:

[0064] Table 5. Moisturizing rate of different samples

[0065]

[0066] The ambient temperature was controlled at 25±2 ℃. A certain amount of glycerol, L-carnosine, sophorolipid, and the L-carnosine-sophorolipid prepared in Example 1 were dissolved in a 50% ethanol aqueous solution at a mass fraction of 1%, and named as the positive control group and experimental groups 1-3, respectively. The 50% ethanol solution served as the blank control group. The above solutions were placed in pre-weighed weighing bottles and placed in a desiccator. The weight of the emerald green was calculated after 1, 2, 4, 8, 12, and 24 hours, with three replicates for each sample. The moisturizing rate % was calculated as (mass after placement / mass before placement) × 100%. The results are shown in Table 5. Compared to the blank control group, experimental groups 1-3 all showed moisturizing effects. The moisturizing effect of L-carnosine was slightly lower than that of glycerol, the moisturizing effect of sophorolipid was comparable to that of glycerol, while the moisturizing effect of L-carnosine-sophorolipid micelles was the best, showing a synergistic moisturizing effect of L-carnosine and sophorolipid. The moisturizing rate could be maintained above 60% within 24 hours.

[0067] In summary, L-carnosine-sophorolipid micelles were successfully prepared using the self-assembly properties of sophorolipids and a thin-film hydration method. These micelles exhibited good carnosinase resistance, improving their stability in vivo. The micellar form also significantly enhanced the transdermal absorption rate of L-carnosine, demonstrating its advantages in topical skincare. Furthermore, the L-carnosine-sophorolipid micelles also possess free radical scavenging, anti-inflammatory, antibacterial, and moisturizing effects, showcasing the synergistic skincare benefits of L-carnosine and sophorolipids.

[0068] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. Those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A method for preparing L-carnosine-sophorolipid micelles, characterized in that, Includes the following steps: Sophorolipid was dissolved in an organic solvent to obtain a sophorolipid solution; L-carnosine was dissolved in water to obtain an L-carnosine aqueous solution; Mix the sophorolipid solution with the L-carnosine aqueous solution; Remove organic solvents to form a thin film; The membrane was hydrated with physiological saline to form a micelle solution; The micelle solution was purified to remove unreacted L-carnosine, and the retentate was lyophilized to obtain L-carnosine-sophorolipid micelles. Organic solvents include methanol, ethanol and / or acetone; The pH value of L-carnosine aqueous solution is 2 to 7; The mass ratio of M-sophorolipid to L-carnosine is 3:1 to 8:1; The step of removing organic solvents is vacuum rotary evaporation at a vacuum level of -0.06 MPa to -0.08 MPa and a temperature of 30°C to 70°C.

2. The method for preparing L-carnosine-sophorolipid micelles according to claim 1, characterized in that, The amount of physiological saline added is 1-1.5 times the volume; the hydration time is 8-12 minutes.

3. The method for preparing L-carnosine-sophorolipid micelles according to claim 1, characterized in that, The purification steps include ultrafiltration using a 1000-1500 Da ultrafiltration membrane.

4. An L-carnosine-sophorolipid micelle, characterized in that, Obtained by the preparation method according to any one of claims 1 to 3.

5. The application of the L-carnosine-sophorolipid micelles according to claim 4 in the preparation of skin care products.

Images