Neohesperidin nano-liposome, sunscreen emulsion as well as preparation method and application of neohesperidin nano-liposome and sunscreen emulsion

Abstract

The invention belongs to the technical field of skin care products, and particularly relates to neohesperidin nano-liposome, a sunscreen emulsion and a preparation method and application of the neohesperidin nano-liposome and the sunscreen emulsion. The neohesperidin nano-liposome provided by the invention is formed by wrapping neohesperidin in a lipid membrane, and in-vitro experiments prove that the emulsion containing the neohesperidin nano-liposome can significantly improve UVB-induced mouse skin light injury, and the skin presents a small amount of injury traces; skin swelling and corium layer thickening induced by UVB irradiation are reduced, corium collagenous fiber reduction caused by UVB irradiation is remarkably improved, and the relative content of collagenous fibers after neohesperidin acts is remarkably increased.

Description

Technical Field

[0001] The present invention belongs to the technical field of skin care products, and more specifically, relates to neohesperidin nanoliposomes, sunscreen lotion, and their preparation methods and applications. Background Art

[0002] Skin photoaging refers to the skin aging phenomenon caused by long-term exposure of the skin to ultraviolet radiation, including external manifestations such as sunburn, wrinkles, surface dryness, rough peeling, photocarcinogenesis, etc. With the increase in ground ultraviolet rays caused by environmental problems, the incidence of skin photoaging diseases and skin diseases such as photosensitive dermatitis caused by them shows an increasing trend year by year, seriously affecting people's quality of life. Ultraviolet rays in the UVB band are the main factors causing acute skin damage. When the skin is strongly irradiated by UVB for a long time, it will induce early and delayed erythema responses. Cells damaged by UVB release histamine and prostaglandins (PGs), and then release interleukins in response to vasodilation and erythema formation. Severe erythema effects may be accompanied by edema, blisters, peeling, skin itching, etc., ultimately leading to the occurrence of skin photoaging. Therefore, timely repairing skin inflammation caused by UV radiation damage is the first step in inhibiting skin photoaging. Effective anti-inflammatory drugs can slow down this delayed photoallergy reaction process, inhibit the inflammatory response, and thus slow down skin photoaging. In view of the many side effects of traditional non-steroidal and steroidal anti-inflammatory drugs, screening anti-photoaging plant components with both anti-inflammatory and antioxidant activities from traditional Chinese medicinal materials or natural products has also been one of the hotspots attracting much attention from researchers in recent years.

[0003] Neohesperidin (NH) is a flavanone glycoside compound widely present in plants such as Mesembryanthemum crystallinum Carpobrotus edulis L.), Citrus aurantium var. amara Citrus changshanensis L . ), Citrus aurantium Citrus aurantium L.), Citrus jambhiri Citrus jambhiri Lush L.), Citrus paradisi Citrus grandis L Osbeck), and Citrus sinensis Citrus sinensis Osbeck), etc., and is a structural analog of hesperidin. Neohesperidin is extracted, separated, and purified from the traditional Chinese medicine Fructus Aurantii Immaturus. Fructus Aurantii Immaturus was first recorded in "Shennong Ben Cao Jing" and is the dried immature fruit of Citrus aurantium Citurs aurantium L.) and its cultivated varieties. Fructus Aurantii Immaturus is rich in various bioactive substances such as flavonoids, volatile oils, alkaloids, and coumarins. Neohesperidin was early used as vitamin P. In recent years, research reports have shown that neohesperidin has various biological activities, such as antidepressant, increasing glucose consumption and AMPK protein expression in HepG2 cells, antioxidant, antitumor, antifungal effects, etc., but there are no reports on its anti-photoaging research. Summary of the Invention

[0004] The purpose of this invention is to provide novel hesperidin nanoliposomes, sunscreen emulsions, their preparation methods, and applications.

[0005] The present invention provides novel hesperidin nanoliposomes, which are formed by encapsulating novel hesperidin in a lipid membrane, and the particle size of the novel hesperidin nanoliposomes is 200~300nm.

[0006] The novel hesperidin nanoliposomes provided by this invention are liposomes formed by encapsulating novel hesperidin in a lipid membrane. Keratinocyte experiments show that novel hesperidin can effectively increase the survival rate of photodamaged cells. Animal skin photoaging models show that novel hesperidin has good anti-UVB-induced photoaging activity and can significantly increase the relative content of collagen fibers.

[0007] This invention also provides a method for preparing novel hesperidin nanoliposomes, the method of which is as follows; Soybean phospholipids, cholesterol, neohesperidin and dichloromethane were mixed and evaporated under reduced pressure to form a film. After removing organic reagents, water was added for hydration and ultrasonic treatment for 10-30 minutes to obtain neohesperidin nanoliposomes. The mass-to-volume ratio of soybean phospholipids, cholesterol, neohesperidin, dichloromethane, and water is 300-400 mg: 60-70 mg: 5-15 mg: 10-20 mL: 45-55 mL.

[0008] Furthermore, the ultrasonic power is 150~250W.

[0009] The present invention also provides a sunscreen lotion, which is made of the following ingredients in weight percentage: 4%~6% of the neohesperidin nanoliposomes as described in claim 1, 2%~4% of aloe polysaccharide, 0.4%~0.6% of high molecular weight sodium hyaluronate, 0.4%~0.6% of low molecular weight sodium hyaluronate, 0.5%~1.5% of vitamin E, 4%~6% of hydrogenated castor oil, 2%~4% of 1,2-pentanediol, 0.4%~0.6% of ethylhexylglycerin, 2%~4% of glycerin, and water to make up 100%.

[0010] The present invention also provides the application of the novel hesperidin nanoliposomes in the preparation of sunscreen lotions.

[0011] Furthermore, the sunscreen lotion is used to increase the collagen fiber content in sun-damaged skin.

[0012] The present invention also provides the application of the novel hesperidin nanoliposomes in the preparation of drugs for treating photodamage to the skin.

[0013] Furthermore, the drug is used to improve the survival rate of keratinocytes after photodamage.

[0014] The present invention has the following beneficial effects: The novel hesperidin nanoliposomes provided by this invention are based on nanotechnology and can effectively repair and improve skin photodamage caused by UVB irradiation. They also have enhanced skin permeability and bioavailability and can be used for the repair and improvement of skin photodamage caused by UVB. Attached Figure Description

[0015] Figure 1 This is a blank liposome particle size distribution image.

[0016] Figure 2 This is a particle size distribution diagram of the new hesperidin nanoliposomes.

[0017] Figure 3 A statistical graph showing the effect of non-liposome-encapsulated neohesperidin on the survival rate of HaCaT cells irradiated by UVB.

[0018] Figure 4 Statistical graph showing the effect of liposome-encapsulated novel hesperidin on the survival rate of HaCaT cells irradiated by UVB.

[0019] Figure 5 The statistical graphs show the effect of liposome-encapsulated neohesperidin on apoptosis of HaCaT cells irradiated by UVB. In the graphs, A is the flow cytometry graph of the blank group, B is the flow cytometry graph of the UVB group, C is the flow cytometry graph of the cells treated with 0.05 mg / mL NH nanoliposomes, D is the flow cytometry graph of the cells treated with 0.1 mg / mL NH nanoliposomes, E is the flow cytometry graph of the cells treated with 0.2 mg / mL nanoliposomes, and F is a statistical graph of apoptosis rate, mortality rate and survival rate of HaCaT cells.

[0020] Figure 6 Histological evaluation of UVB-irradiated mouse skin by a sunscreen lotion containing neohesperidin.

[0021] Figure 7 This is a statistical graph showing the effect of neohesperidin nanoliposome emulsion on the thickness of the dermal layer of mouse skin.

[0022] Figure 8 This is a statistical graph showing the effect of neohesperidin nanoliposome emulsion on the relative content of collagen fibers in the dermal layer of mouse skin. Detailed Implementation

[0023] The present invention will now be described in detail with reference to specific embodiments, but these should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments can be obtained commercially unless otherwise specified.

[0024] Example 1: Preparation and particle size determination of neohesperidin nanoliposomes.

[0025] Liposomes containing neohesperidin were prepared using a thin-film ultrasonic method. 337.5 mg of soybean lecithin was dissolved in 11.25 mL of dichloromethane to obtain a soybean lecithin solution, and 65 mg of cholesterol was dissolved in 3.25 mL of dichloromethane to obtain a cholesterol solution. 10 mg, 5 mg, and 2.5 mg of neohesperidin were dissolved in 1 mL of dichloromethane to obtain neohesperidin solutions of different concentrations. The three different concentrations of neohesperidin solutions were poured into a round-bottom flask, and the soybean lecithin and cholesterol solutions were added. After thorough mixing, the mixture was rotary evaporated under reduced pressure to form a thin film. The organic reagents were removed, and the mixture was then placed in a fume hood until all organic reagents had evaporated. 50 mL of ultrapure water was added for hydration, and the mixture was then sonicated for 20 min in an ultrasonic instrument with a power of 200 W. This yielded neohesperidin nanoliposomes containing 0.2 mg / mL, 0.1 mg / mL, and 0.05 mg / mL, respectively. Blank liposomes were prepared using the same procedure except that neohesperidin was not added. In the cell viability evaluation experiment, MEM basal medium was used instead of ultrapure water for hydration.

[0026] The particle size of liposomes was determined using a nanoparticle size analyzer. The results are as follows: Figure 1 and picture Figure 2 It can be seen that the particle size of the blank liposomes is about 185.8 nm, and the particle size of the nanoliposomes after encapsulating neohesperidin is about 284 nm.

[0027] Example 2: Effects of novel hesperidin nanoliposomes on UVB-irradiated cells.

[0028] 1. Effect of neohesperidin nanoliposomes on the viability of UVB-irradiated HaCaT cells HaCaT cells were seeded into 96-well plates when they reached approximately 80% confluence, with a cell density of 5 × 10⁶ cells / well. 5Cells / mL. Plated cells were used for experiments the following day. Experimental groups were: Control group, UVB model group, UVB + 0.05 mg / mL neohesperidin nanoliposome group, UVB + 0.1 mg / mL neohesperidin nanoliposome group, UVB + 0.2 mg / mL neohesperidin nanoliposome group, and UVB + blank liposome group. After incubating cells with neohesperidin liposomes for 1 hour, UVB irradiation was performed. 33 μL of culture medium was reserved in each well to prevent cell drying during irradiation. UVB irradiation doses were 30, 60, 90, and 120 mJ / cm². After irradiation, fresh culture medium was added to each well to a total of 100 μL. After 24 hours of culture, the medium was replaced with medium containing 10% CCK-8 (v / v) and incubated at 37°C for 1 hour. The absorbance at 450 nm was measured using a microplate reader, and the relative cell viability was calculated. Simultaneously, the same experiment was performed using unencapsulated neohesperidin (0.05 mg / mL, 0.1 mg / mL, 0.2 mg / mL) to determine cell viability.

[0029] Experimental results: such as Figure 3 and 4 As shown, UVB irradiation significantly reduced cell viability, and this reduction was negatively correlated with the irradiation dose. Different concentrations of neohesperidin improved cell viability, and this improvement was positively correlated with the concentration of neohesperidin. (Using UVB at 90 mJ / cm² as an example...) 2 Taking the dosage groups as an example, the cell survival rate in the UVB group was only 19.74%, while the cell survival rates in the 0.05 mg / mL, 0.1 mg / mL, and 0.2 mg / mL neohesperidin groups were 23.75%, 29.57%, and 38.81%, respectively. Intervention with neohesperidin nanoliposomes significantly improved the cell survival rate in all UVB dosage groups (90 mJ / cm²). 2 At concentrations of 0.05 mg / mL, 0.1 mg / mL, and 0.2 mg / mL, the cell viability of neohesperidin nanoliposomes was 54.44%, 58.85%, and 63.90%, respectively, which were significantly higher than those of the neohesperidin group. This indicates that neohesperidin nanoliposomes have a significant inhibitory effect on UVB radiation-induced photodamage to epidermal cells.

[0030] 2. Effects of neohesperidin nanoliposomes on apoptosis of UVB-irradiated HaCaT cells HaCaT cells were seeded into 6-well plates when they reached approximately 80% confluence, with a cell density of 5 × 10⁶ cells / well. 5The concentration of nanoparticles per mL was 90 mJ / mL. The following day, the groups were: Control group, UVB model group, UVB + 0.05 mg / mL neohesperidin nanoliposome group, UVB + 0.1 mg / mL neohesperidin nanoliposome group, and UVB + 0.2 mg / mL neohesperidin nanoliposome group. After incubation with 0.05, 0.1, and 0.2 mg / mL neohesperidin nanoliposomes for 1 hour, the concentration was 90 mJ / cm². 2 Irradiate. After 6 hours, digest and collect cells (centrifuge at 1000 rpm, 4°C for 10 min). Add 1 mL of pre-chilled PBS to the pellet, gently vortex to resuspend the cells, and centrifuge again (1000 rpm, 4°C for 10 min), discarding the supernatant. Resuspend the cells in 200 μL buffer, add 10 μL of L-Annexin V-FITC and 10 μL of PI, mix gently, incubate at room temperature in the dark for 15 min, then add 300 μL buffer and analyze using flow cytometry.

[0031] Experimental results are as follows Figure 5 As shown, UVB irradiation induced a significant decrease in the number of viable HaCaT cells and a significant increase in the number of apoptotic cells. However, different concentrations of neohesperidin nanoliposomes significantly inhibited cell apoptosis and death rates in a dose-dependent manner. That is, as the concentration of neohesperidin increased, the cell apoptosis rate decreased significantly and the number of viable cells increased significantly.

[0032] Example 3: Formulation and preparation process of sunscreen lotion containing neohesperidin.

[0033] 1. Formula, by mass fraction: 0.2 mg / mL neohesperidin nanoliposomes: 5%.

[0034] Aloe polysaccharides: 3%.

[0035] High molecular weight sodium hyaluronate (800,000 to 1,200,000 Da): 0.5%.

[0036] Low molecular weight sodium hyaluronate (200,000~400,000 Da): 0.5%.

[0037] Vitamin E: 1%.

[0038] Hydrogenated castor oil: 5%.

[0039] 1,2-Pentanediol: 3%.

[0040] Ethylhexylglycerin: 0.5%.

[0041] Glycerin: 3%.

[0042] Aloe polysaccharides, high molecular weight sodium hyaluronate, and low molecular weight sodium hyaluronate were purchased from Shaanxi Yuanshengte Biotechnology Co., Ltd.

[0043] 2. Raw material preparation (calculated based on 200g of finished product) 10 mL of hydrogenated castor oil (emulsifier).

[0044] Vitamin E is 2 mL.

[0045] 10g of 0.2mg / mL neohesperidin nanoliposomes.

[0046] 6g of aloe polysaccharides.

[0047] 1g of high molecular weight sodium hyaluronate (pre-dissolved in 50mL of 50℃ water).

[0048] 1g of low molecular weight sodium hyaluronate (pre-dissolved in 50mL of room temperature water).

[0049] 1,2-Pentanediol (preservative + moisturizer) 6g.

[0050] Ethylhexylglycerin (preservative aid) 1g.

[0051] Glycerin (to enhance moisturizing properties), 4.8 mL dissolved in 5 mL of pure water.

[0052] Add deionized water to bring the total to 200g.

[0053] 3. Operating steps: Oil phase preparation: Mix 10 mL of hydrogenated castor oil, 2 mL of vitamin E, and 6 g of glycerin, and heat to 75 °C until completely dissolved.

[0054] Aqueous phase preparation: Dissolve 6g of aloe polysaccharide, 6g of 1,2-pentanediol, and 1g of ethylhexylglycerin in about 80mL of deionized water and heat to 75℃.

[0055] Stepwise dissolution of sodium hyaluronate: Pre-disperse 1g of high molecular weight sodium hyaluronate in a small amount of water, then add it to warm water (50℃), bringing the total water volume to 50mL. Stir until a gel-like consistency is reached. Dissolve 1g of low molecular weight sodium hyaluronate directly in 50mL of room temperature water and stir until transparent.

[0056] Emulsification process: Slowly pour the oil phase into the aqueous phase and stir in a homogenizer (3000~5000rpm) for 3~5min until a homogeneous emulsion is formed. When the temperature drops to 40℃, add about 100mL of pre-dissolved sodium hyaluronate solution and 10g of 0.2mg / mL neohesperidin liposomes, and stir at low speed to avoid demulsification.

[0057] Cooling and adjustment: After cooling to 30℃, the pH was measured and adjusted to 6.5~7.0 with citric acid. The product was then filled into light-proof aseptic containers to obtain a sunscreen lotion containing neohesperidin.

[0058] 4. Evaluation of the anti-photodamage activity of emulsions containing neohesperidin C57BL / 6 mice (male, 7 weeks old, 23±2g) were housed individually and acclimatized for one week (ambient humidity 40-60%, temperature 24℃, light / dark cycle 12 / 12h). Afterward, they were randomly divided into three groups of 10 mice each: a control group, a UVB model group, and a group containing neohesperidin emulsion. The mice were housed for one day after hair removal on their backs. The UVB group involved exposing the bare skin to a UVB concentration of 100 mJ / cm². 2 Under UVB (312nm), the irradiation intensity is 167w / cm². 2 The irradiation time was 10 minutes, repeated every 24 hours for a total of two times. The Ctr group received no treatment, and the irradiation source was provided by a 312nm ultraviolet lamp. The irradiation intensity was measured by a Xinbao Scientific UV340B ultraviolet detector. When scales, erythema, wrinkles, and even ulceration, scabs, and a leathery feel appeared on the irradiated area of ​​the mouse's back, it indicated that the UVB radiation-induced mouse skin damage model was successfully established.

[0059] In the NH-NLS group, mice were irradiated with a neohesperidin emulsion on their bare skin for 0.5 hours, followed by UVB irradiation twice, every 24 hours, at a dose of 100 mJ / cm². 2 Twenty-four hours after the second irradiation, the skin morphology of the mice was photographed and histologically evaluated using H&E staining and Masson staining. The results are as follows: Experimental results are as follows Figure 6 , Figure 7 and Figure 8 As shown, compared with normal animal skin, UVB irradiation caused severe photodamage, manifested as skin swelling, crusting, and lesions. Intervention with neohesperidin liposomes significantly improved skin damage, bringing the skin condition close to that of normal skin. This phenomenon was verified by histological staining. H&E results showed that the dermal layer thickness of the UVB-irradiated model skin was significantly increased, the epidermis was significantly thickened, and the skin structure was disordered with irregular cell arrangement. Intervention with neohesperidin liposomes significantly reduced dermal layer thickness, and the cells were arranged more neatly. Masson staining results also showed that after UVB irradiation, collagen fibers in the dermal layer of the skin exhibited significant breakage, dissolution, and sparse arrangement, indicating that UVB irradiation led to severe collagen degradation and matrix destruction. Intervention with neohesperidin nanoliposomes significantly improved the breakage and dissolution of collagen fibers in the dermal layer of the skin, effectively restoring fiber density and integrity.

[0060] The above results show that emulsions containing neohesperidin nanoliposomes can significantly improve UVB-induced photodamage to mouse skin, with the skin showing only minor damage marks; reduce UVB-induced skin swelling and dermal thickening; significantly improve the reduction of dermal collagen fibers caused by UVB irradiation; and significantly increase the relative content of collagen fibers after neohesperidin treatment.

[0061] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0062] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of the invention.

[0063] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A novel hesperidin nanoliposome, characterized in that, The neohesperidin nanoliposomes are formed by encapsulating neohesperidin in a lipid membrane, and the particle size of the neohesperidin nanoliposomes is 200~300nm.

2. The method for preparing the novel hesperidin nanoliposomes according to claim 1, characterized in that, The preparation method is as follows: soybean phospholipids, cholesterol, neohesperidin and dichloromethane are mixed, and the mixture is evaporated under reduced pressure to form a thin film. After removing the organic reagents, water is added for hydration, and the mixture is ultrasonically treated for 10-30 minutes to obtain neohesperidin nanoliposomes. The mass-to-volume ratio of soybean phospholipids, cholesterol, neohesperidin, dichloromethane, and water is 300-400 mg: 60-70 mg: 5-15 mg: 10-20 mL: 45-55 mL.

3. The preparation method according to claim 2, characterized in that, The ultrasonic power is 150~250W.

4. A sunscreen lotion, characterized in that, The sunscreen lotion is made from the following ingredients in the indicated weight percentages: 4%~6% of the neohesperidin nanoliposomes as described in claim 1, 2%~4% of aloe polysaccharide, 0.4%~0.6% of high molecular weight sodium hyaluronate, 0.4%~0.6% of low molecular weight sodium hyaluronate, 0.5%~1.5% of vitamin E, 4%~6% of hydrogenated castor oil, 2%~4% of 1,2-pentanediol, 0.4%~0.6% of ethylhexylglycerin, 2%~4% of glycerin, and water to make up 100%.

5. The application of the novel hesperidin nanoliposomes according to claim 1 in the preparation of sunscreen emulsions.

6. The application according to claim 5, characterized in that, The sunscreen lotion is used to increase the collagen fiber content in sun-damaged skin.

7. The use of the novel hesperidin nanoliposomes of claim 1 in the preparation of drugs for treating photodamage to the skin.

8. The application according to claim 7, characterized in that, The drug is used to improve the survival rate of keratinocytes after photodamage.

Images