Exocarpium citri grandis polyphenol hybrid liposome and preparation method thereof as well as skin lotion and preparation method thereof

Abstract

The invention relates to exocarpium citri grandis polyphenol hybrid liposome and a preparation method thereof as well as skin lotion and a preparation method thereof. The exocarpium citri grandis polyphenol hybrid liposome comprises phospholipid, cholesterol, a cell penetrating factor and exocarpium citri grandis polyphenol, the weight ratio of the phospholipid to the cholesterol to the cell penetrating factor to the pummelo peel polyphenol is 10: (0.5-3): (0.5-8): (0.1-6). The exocarpium citri grandis polyphenol is wrapped in a liposome structure, the application effect of the exocarpium citri grandis polyphenol compound in a water-based skin care product is effectively improved, the exocarpium citri grandis polyphenol hybrid liposome can show multiple effects of moisturizing, diminishing inflammation, resisting oxidation and the like when being applied to skin moisturizing water, and the exocarpium citri grandis polyphenol hybrid liposome has wide market prospects and practical application value.

Description

Technical Field

[0001] The present disclosure relates to the technical field of daily chemical products, and in particular to a tangerine peel polyphenol hybrid liposome and a preparation method thereof, a lotion and a preparation method thereof. Background Art

[0002] Huajuhong is the immature or nearly mature outer peel or fruit of Huazhou pomelo of the genus Citrus in the Rutaceae family. It is mainly produced in Huazhou, Guangdong, China, and is widely used due to its specific medicinal value. Huajuhong is traditionally used as an oral Chinese medicine or food, and has the effects of regulating qi and relieving fullness, dispelling cold and relieving cough, and strengthening the spleen and digestion. Modern research has found that Huajuhong is rich in a variety of bioactive ingredients, especially polyphenol compounds (such as naringin, wild sumac glycosides, rutin naringin and naringenin, etc.), which show significant effects in anti-inflammatory and antioxidant aspects. However, the poor water solubility, easy degradation, and low bioavailability of Huajuhong polyphenols limit their application and efficacy in water-based products. Summary of the invention

[0003] The purpose of the present invention is to provide a citrus aurantium polyphenol hybrid liposome for anti-inflammatory lotion and a preparation method thereof, lotion and a preparation method thereof.

[0004] In order to achieve the above-mentioned purpose, the first aspect of the present disclosure provides a tangerine peel polyphenol hybrid liposome, which includes phospholipids, cholesterol, cell penetrating factors and tangerine peel polyphenol extract; the weight ratio of the phospholipids, the cholesterol, the cell penetrating factors and the tangerine peel polyphenol extract is 10: (0.5~3): (0.5~8): (0.1~6).

[0005] Optionally, the phospholipids include one or more of sunflower seed lecithin, soybean lecithin and sphingomyelin.

[0006] Optionally, the cell penetrating factor comprises one or more of phospholipid-polyethylene glycol-octaarginine, phospholipid-polyethylene glycol-nonaarginine and phospholipid-polyethylene glycol-cell penetrating peptide.

[0007] In a second aspect of the present disclosure, a method for preparing the tangerine peel polyphenol hybrid liposomes described in the first aspect of the present disclosure is provided, the method comprising:

[0008] The tangerine peel polyphenol extract, phospholipids, cholesterol, cell penetrating factor and solvent are stirred and mixed to obtain a mixed solution;

[0009] The mixed solution is subjected to reduced pressure rotary evaporation to obtain a lipid film;

[0010] The lipid film is mixed with the first sterile water, and then subjected to ultrasonic dispersion treatment, and then freeze-dried to obtain the tangerine peel polyphenol hybrid liposome.

[0011] Optionally, the solvent is ethanol and chloroform, and the weight ratio of the phospholipid, the ethanol and the chloroform is 1:(14-34):(7-17); and / or,

[0012] The stirring and mixing conditions include: a stirring speed of 100 to 500 rpm.

[0013] Optionally, the conditions of the reduced pressure rotary evaporation include: vacuum degree of 0.01-0.10 MPa, temperature of 45-60° C., and rotation speed of 50-200 rpm; and / or,

[0014] The conditions of the ultrasonic dispersion treatment include: ultrasonic power of 400-800W and time of 3-8min.

[0015] According to a third aspect of the present disclosure, a lotion is provided, wherein the lotion comprises the tangerine peel polyphenol hybrid liposomes described in the first aspect of the present disclosure; based on the total weight of the lotion, the lotion comprises 2.8 to 8.2 weight % of the tangerine peel polyphenol hybrid liposomes, 0.01 to 0.4 weight % of a thickener, 0.12 to 2.3 weight % of a humectant, 0.01 to 0.75 weight % of an antibacterial agent and the remainder water.

[0016] Optionally, the thickener includes one or more of β-glucan, flaxseed gum and tremella polysaccharide.

[0017] Optionally, the moisturizer includes one or more of sodium pyrrolidonecarboxylate, potassium pyrrolidonecarboxylate, L-serine, hyaluronic acid and ceramide.

[0018] Optionally, the antibacterial agent includes one or more of potassium cinnamate, epigallocatechin gallate and tannic acid.

[0019] In a fourth aspect of the present disclosure, a method for preparing the lotion according to the third aspect of the present disclosure is provided, the method comprising:

[0020] mixing the thickener with the second sterile water to obtain a thickener solution;

[0021] Mixing the tangerine peel polyphenol hybrid liposome with the third sterile water to obtain a tangerine peel polyphenol hybrid liposome solution;

[0022] The thickener solution, the citrus aurant polyphenol hybrid liposome solution, the moisturizer and the antibacterial agent are mixed and then vacuum stirred to obtain a lotion.

[0023] Optionally, the vacuum stirring conditions include: a stirring speed of 1500 to 6000 rpm and a stirring time of 10 to 60 min.

[0024] Through the above technical scheme, the present invention encapsulates the tangerine peel polyphenol extract in a liposome structure, which can significantly improve its dispersibility and stability in a water-based system. At the same time, by introducing cell penetration factors into the liposomes, the liposomes can more effectively penetrate the skin barrier layer, thereby improving the absorption efficiency and anti-inflammatory activity of tangerine peel polyphenol compounds, and effectively improving the application effect of tangerine peel polyphenol compounds in water-based skin care products. The tangerine peel polyphenol hybrid liposomes used in lotions can exhibit multiple effects such as moisturizing, anti-inflammatory, and anti-oxidation, and have broad market prospects and practical application value.

[0025] Other features and advantages of the present disclosure will be described in detail in the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The accompanying drawings are used to provide a further understanding of the present disclosure and constitute a part of the specification. Together with the following specific embodiments, they are used to explain the present disclosure but do not constitute a limitation of the present disclosure. In the accompanying drawings:

[0027] Figure 1 is a schematic diagram of the structure of tangerine peel polyphenol hybrid liposomes in a specific embodiment;

[0028] Figure 2 is the composition of the tangerine peel polyphenol extract used in the examples and the mass spectrum of its main substance content;

[0029] Figure 3 is a microscopic structural photograph of the tangerine peel polyphenol hybrid liposome prepared in Example 1;

[0030] Figure 4 This is the evaluation result of the cellular uptake of the tangerine peel polyphenol hybrid liposome prepared in Example 3. DETAILED DESCRIPTION

[0031] The specific implementation of the present disclosure is described in detail below in conjunction with the accompanying drawings. It should be understood that the specific implementation described herein is only used to illustrate and explain the present disclosure, and is not used to limit the present disclosure.

[0032] In a first aspect, the present disclosure provides a tangerine peel polyphenol hybrid liposome, which comprises phospholipids, cholesterol, a cell penetrating factor and a tangerine peel polyphenol extract; the weight ratio of the phospholipids, the cholesterol, the cell penetrating factor and the tangerine peel polyphenol extract is 10: (0.5-3): (0.5-8): (0.1-6).

[0033] The tangerine peel polyphenol hybrid liposome disclosed in the present invention has a spherical vesicle structure. Figure 1As shown, the double-layer vesicles formed by the arrangement of phospholipid molecules can effectively encapsulate the tangerine peel polyphenol extract, so that it maintains a high stability in the aqueous phase system and prevents its degradation in the aqueous environment; hybrid liposomes refer to liposome structures embedded with functional compounds. In the present disclosure, by embedding cell penetrating factors in the double-layer vesicle structure, the cell membrane penetration ability of the liposome can be improved, so that the tangerine peel polyphenol compounds can be more efficiently absorbed by the skin, thereby significantly improving the bioavailability and anti-inflammatory activity of the tangerine peel polyphenol compounds.

[0034] By controlling the ratio of each component, it is beneficial to improve the stability and anti-inflammatory activity of the citrus red polyphenol hybrid liposome. In a preferred embodiment, the weight ratio of the phospholipid, the cholesterol, the cell penetrating factor, and the citrus red polyphenol extract is 10: (1-2): (1-5): (1-3).

[0035] In the present disclosure, the main components of the tangerine peel polyphenol extract include naringin (CAS No. 14259-46-2), naringin (CAS No. 10236-47-2), rhoifolin (CAS No. 17306-46-6) and naringenin (CAS No. 480-41-1), and the total content of the above four components accounts for more than 75% by weight of the dry weight of the tangerine peel polyphenol extract. The content of each component can be adjusted within a certain range. Specifically, based on the dry weight of the tangerine peel polyphenol extract, the content of naringin can be 0.4-0.9% by weight, the content of naringin can be 69.5-85.5% by weight, the content of rhoifolin can be 2.8-4.5% by weight, and the content of naringenin can be 3.2-5.5% by weight.

[0036] In one embodiment, the phospholipids may include one or more of sunflower seed phospholipids, soybean lecithin and sphingomyelin. The above phospholipids exhibit excellent membrane structure stability and biocompatibility, which is beneficial to significantly improve the stability of the liposome structure in the aqueous phase and enhance the penetration and absorption of tangerine peel polyphenols in the skin.

[0037] In one embodiment, the cell penetrating factor may include one or more of phospholipid-polyethylene glycol-octaarginine, phospholipid-polyethylene glycol-nonaarginine and phospholipid-polyethylene glycol-cell penetrating peptide. Among them, octaarginine (Arg8) has good hydrophilicity and is positively charged, and can interact with the cell membrane to penetrate the cell membrane and enter the cell; nonaarginine (Arg9) has similar performance to octaarginine, and also has neuroprotective function; cell penetrating peptide (TAT) is a short peptide composed of tyrosine, glycine, arginine, lysine and glutamine, which can enter the cell through a non-specific pathway and deliver its connected substance into the cell; the above-mentioned complex exhibits excellent cell penetrating performance, can carry liposomes to penetrate the cell membrane and enter the cell, and significantly improve the bioavailability of tangerine red polyphenols.

[0038] According to the present disclosure, the tangerine peel polyphenol hybrid liposome has a smaller particle size, showing good dispersibility and skin absorption. Specifically, the particle size of the tangerine peel polyphenol hybrid liposome can be 60 to 150 nm, preferably 76 to 95 nm.

[0039] In a second aspect of the present disclosure, a method for preparing the tangerine peel polyphenol hybrid liposomes described in the first aspect of the present disclosure is provided, the method comprising the following steps S101 to S103:

[0040] S101, stirring and mixing the tangerine peel polyphenol extract, phospholipids, cholesterol, cell penetrating factor and solvent to obtain a mixed solution;

[0041] S102, performing vacuum rotary evaporation on the mixed solution to obtain a lipid film;

[0042] S103, mixing the lipid film with the first sterile water, performing ultrasonic dispersion treatment, and then freeze-drying to obtain the tangerine peel polyphenol hybrid liposomes.

[0043] In step S101, the polyphenol extract of Citrus aurantium can be obtained by a common extraction method in the art. In one embodiment, the extraction step of the polyphenol extract of Citrus aurantium can include:

[0044] The tangerine peel was crushed and passed through a 50-200 mesh sieve, 50-120 g of tangerine peel powder was weighed and added with 70-95 wt % ethanol at a weight ratio of 1:5, and constant temperature ultrasonic-assisted ethanol extraction was performed at an ultrasonic power of 160-240 W and 25° C. for 1 h, and the extraction was repeated three times and concentrated under reduced pressure to obtain a crude extract of tangerine peel polyphenols;

[0045] The crude extract of tangerine peel polyphenol is defatted with petroleum ether, purified with AB-8 macroporous adsorption resin, eluted with 0-15 wt% methanol to remove water-soluble impurities of polysaccharides, eluted with 80 wt% methanol and collected, and after rotary evaporation to remove methanol, freeze-dried to obtain the tangerine peel polyphenol extract.

[0046] The specific types of the phospholipids and cell penetrating factors are described in the first aspect of the present disclosure. The solvent can be an organic solvent commonly used in the art, and its amount can be adjusted within a certain range. In a specific embodiment, the solvent is ethanol and chloroform, and the weight ratio of the phospholipids, the ethanol and the chloroform can be 1: (14-34): (7-17). The stirring and mixing conditions can include: the stirring speed is 100-500rpm.

[0047] In step S102, the conditions for the reduced pressure rotary evaporation may include: a vacuum degree of 0.01 to 0.10 MPa, a temperature of 45 to 60°C, and a rotation speed of 50 to 200 rpm.

[0048] In step S103, the amount of the first sterile water can be adjusted as needed. For example, the weight ratio of the lipid film to the first sterile water can be 1:(30-100); the conditions for the ultrasonic dispersion treatment may include: ultrasonic power of 400-800 W and time of 3-8 min.

[0049] The present invention discloses a method of preparing stable tangerine peel polyphenol hybrid liposomes by combining thin-film hydration method with ultrasonic dispersion technology, thereby improving the bioavailability and anti-inflammatory activity of tangerine peel polyphenol compounds. It can exhibit excellent moisturizing, anti-inflammatory, antioxidant and other multiple effects in skin care applications, and has broad market prospects and practical application value.

[0050] In a third aspect of the present disclosure, a lotion is provided, comprising the tangerine peel polyphenol hybrid liposomes described in the first aspect of the present disclosure; based on the total weight of the lotion, the lotion comprises 2.8 to 8.2 weight % of the tangerine peel polyphenol hybrid liposomes, 0.01 to 0.4 weight % of a thickener, 0.12 to 2.3 weight % of a humectant, 0.01 to 0.75 weight % of an antibacterial agent and the remainder water.

[0051] The present invention uses tangerine peel polyphenol hybrid liposome as the core active ingredient, optimizes the formula, and combines thickener, moisturizer and antibacterial agent components to obtain an anti-inflammatory lotion, in which the tangerine peel polyphenol active ingredient can act deeply on the skin, exert lasting anti-inflammatory and antioxidant effects, and effectively lock in moisture, provide long-lasting moisturizing, and keep the skin hydrated and smooth.

[0052] In a preferred embodiment, based on the total weight of the lotion, the lotion includes 4.7 to 5.6 weight % of the tangerine peel polyphenol hybrid liposome, 0.10 to 0.32 weight % of a thickener, 0.35 to 1.25 weight % of a humectant, 0.08 to 0.35 weight % of an antibacterial agent and the remainder of water.

[0053] In one embodiment, the thickener may include one or more of β-glucan, flaxseed gum and tremella polysaccharide. The above thickeners are beneficial to improving the texture and viscosity of the lotion product and enhancing the user experience.

[0054] In one embodiment, the moisturizer may include one or more of sodium pyrrolidonecarboxylate, potassium pyrrolidonecarboxylate, L-serine, hyaluronic acid and ceramide. The above moisturizers can significantly enhance the moisturizing and water-locking effect of lotion products, increase skin elasticity, and keep the skin hydrated and shiny.

[0055] In one embodiment, the antibacterial agent may include one or more of potassium cinnamate, epigallocatechin gallate and tannic acid. The antibacterial agent can impart good antibacterial properties to the lotion product and extend the service life of the product.

[0056] The lotion disclosed in the present invention can be used as a multi-functional product, which can effectively simplify the skin care steps and avoid the discomfort caused by the inability to integrate skin care products due to the superposition of multiple products. It is widely applicable to people with various skin types and has broad market application prospects.

[0057] In a fourth aspect of the present disclosure, a method for preparing the lotion according to the third aspect of the present disclosure is provided, the method comprising the following steps S110 to S130:

[0058] S110, mixing the thickener with the second sterile water to obtain a thickener solution;

[0059] S120, mixing the tangerine peel polyphenol hybrid liposome with the third sterile water to obtain a tangerine peel polyphenol hybrid liposome solution;

[0060] S130, mixing the thickener solution, the tangerine peel polyphenol hybrid liposome solution, the moisturizer and the antibacterial agent, and then stirring in vacuum to obtain a lotion.

[0061] In step S110, the specific type of the thickener is as described in the third aspect of the present disclosure. The amount of the second sterile water can be adjusted as needed, specifically, the weight ratio of the thickener to the second sterile water can be 1: (25-80).

[0062] In step S120, the amount of the third sterile water can be adjusted as needed. Specifically, the weight ratio of the tangerine peel polyphenol hybrid liposome to the third sterile water can be 1: (11-31).

[0063] In step S130, the specific types of the moisturizer and antibacterial agent are described in the third aspect of the present disclosure. The vacuum stirring conditions may include: vacuum degree of -0.05MPa to 0.09MPa, stirring speed of 1500 to 6000rpm, and stirring time of 10 to 60min.

[0064] The present disclosure is further described below in conjunction with embodiments, but the present disclosure is not limited to the scope of the following embodiments.

[0065] In the following embodiments, the preparation steps of the polyphenol extract of tangerine peel are as follows: crush tangerine peel through a 50-200 mesh sieve, weigh 50-120g tangerine peel powder, add 70-95% by weight ethanol according to a weight ratio of 1:5, perform constant temperature ultrasonic assisted ethanol extraction for 1h under the conditions of ultrasonic power of 160-240W and 25°C, repeat the extraction three times and combine and concentrate under reduced pressure to obtain a crude extract of tangerine peel polyphenol; after the crude extract of tangerine peel polyphenol is defatted with petroleum ether, it is purified with AB-8 macroporous adsorption resin, eluted with 0-15% by weight methanol to remove water-soluble impurities of polysaccharides, and then eluted with 80% by weight methanol and collected tangerine peel polyphenol, after rotary evaporation to remove methanol, freeze-dried to obtain a tangerine peel polyphenol extract, whose mass spectrum total ion flow chart is as shown Figure 2 As shown, 1 is naringin (content 5.6 mg / g d.w.), 2 is naringin (content 785.8 mg / g dw), 3 is rhusin (content 35.2 mg / g d.w.), and 4 is naringenin (content 38.7 mg / g dw), which together account for more than 75% by weight of the dry matter of the tangerine peel polyphenol extract.

[0066] The remaining raw materials are commercially available products.

[0067] Examples 1 to 5 are used to illustrate the tangerine peel polyphenol hybrid liposomes disclosed herein.

[0068] Example 1

[0069] This embodiment is a tangerine peel polyphenol hybrid liposome, which is prepared from the following components: sunflower seed lecithin, cholesterol, phospholipid-polyethylene glycol-cell penetrating peptide and tangerine peel polyphenol extract, with a weight ratio of 10:2:5:3, and the preparation method is as follows:

[0070] Accurately weigh phospholipids, cholesterol, cell penetrating factor and tangerine peel polyphenol extract and put them into a beaker, pour a mixture of ethanol and chloroform (the weight ratio of phospholipids to ethanol and chloroform is 1:20:10) into the beaker, and stir magnetically at 300 rpm at room temperature to completely dissolve the materials to obtain a mixed solution; pour the mixed solution into a round-bottom rotary evaporator, and perform reduced pressure rotary evaporation under the conditions of vacuum degree of 0.05 MPa, temperature controlled at 55°C, and rotation speed of 100 rpm to remove the solvent, forming a smooth lipid film on the wall of the round-bottom flask; add sterile water (the weight ratio of lipid film to sterile water is 1:80) to the round-bottom flask for hydration to obtain a crude liposome solution, and then perform ultrasonic dispersion treatment (ultrasonic power: 800 W, ultrasonic time: 5 min), and freeze-drying to obtain tangerine peel polyphenol hybrid liposomes.

[0071] Atomic force microscopy (AFM) was used to examine the microstructure of the tangerine peel polyphenol hybrid liposomes. Figure 3 As shown, it can be seen that the tangerine peel polyphenol hybrid liposome is a spherical structure with a smooth surface and a particle size of 89.7±0.42nm.

[0072] Example 2

[0073] This embodiment is a hybrid liposome of tangerine peel polyphenols, which is prepared from the following components: soybean lecithin, cholesterol, phospholipid-polyethylene glycol-nona-arginine and tangerine peel polyphenols extract, with a weight ratio of 10:1:2:2, and the preparation method is as follows:

[0074] Accurately weigh phospholipids, cholesterol, cell penetrating factor and tangerine peel polyphenol extract and put them into a beaker, pour a mixture of ethanol and chloroform (the weight ratio of phospholipids to ethanol and chloroform is 1:34:17) into the beaker, and stir magnetically at 500 rpm at room temperature to completely dissolve the materials to obtain a mixed solution; pour the mixed solution into a round-bottom rotary evaporator, and perform reduced pressure rotary evaporation under the conditions of vacuum degree of 0.10 MPa, temperature controlled at 60°C, and rotation speed of 150 rpm to remove the solvent, forming a smooth lipid film on the wall of the round-bottom flask; add sterile water (the weight ratio of lipid film to sterile water is 1:50) to the round-bottom flask for hydration to obtain a crude liposome solution, and then perform ultrasonic dispersion treatment (ultrasonic power: 800 W, ultrasonic time: 3 min), and freeze-drying to obtain tangerine peel polyphenol hybrid liposomes.

[0075] The particle size of the tangerine peel polyphenol hybrid liposomes prepared in this example is 83.3±0.57 nm.

[0076] Example 3

[0077] This embodiment is a hybrid liposome of tangerine peel polyphenols, which is prepared from the following components: sphingomyelin, cholesterol, phospholipid-polyethylene glycol-octaarginine and tangerine peel polyphenols extract, with a weight ratio of 10:2:1:1, and the preparation method is as follows:

[0078] Accurately weigh phospholipids, cholesterol, cell penetrating factor and tangerine peel polyphenol extract and put them in a beaker, pour a mixture of ethanol and chloroform (the weight ratio of phospholipids to ethanol and chloroform is 1:14:7) into the beaker, and stir magnetically at 100 rpm at room temperature to completely dissolve the materials to obtain a mixed solution; pour the mixed solution into a round-bottom rotary evaporator, and perform reduced pressure rotary evaporation under the conditions of vacuum degree 0.01 MPa, temperature control at 45°C, and rotation speed 80 rpm to remove the solvent, forming a smooth lipid film on the wall of the round-bottom flask; add sterile water (the weight ratio of lipid film to sterile water is 1:100) to the round-bottom flask for hydration to obtain a crude liposome solution, and then perform ultrasonic dispersion treatment (ultrasonic power: 400 W, ultrasonic time: 8 min), and freeze-drying to obtain tangerine peel polyphenol hybrid liposomes.

[0079] The particle size of the tangerine peel polyphenol hybrid liposomes prepared in this example is 76.6±0.35 nm.

[0080] Example 4

[0081] The tangerine peel polyphenol hybrid liposomes were prepared according to the method of Example 1, except that the tangerine peel polyphenol hybrid liposomes of this example were prepared from the following components: sunflower seed phospholipids, cholesterol, phospholipid-polyethylene glycol-cell penetrating peptide and tangerine peel polyphenol extract, with a weight ratio of 10:3:8:6.

[0082] The particle size of the tangerine peel polyphenol hybrid liposomes prepared in this example is 147±1.28 nm.

[0083] Example 5

[0084] The tangerine peel polyphenol hybrid liposomes were prepared according to the method of Example 1, except that the tangerine peel polyphenol hybrid liposomes of this example were prepared from the following components: sunflower seed phospholipids, cholesterol, phospholipid-polyethylene glycol-cell penetrating peptide and tangerine peel polyphenol extract, with a weight ratio of 10:0.5:0.5:0.1.

[0085] The particle size of the tangerine peel polyphenol hybrid liposomes prepared in this example is 65±0.23 nm.

[0086] Comparative Example 1

[0087] The tangerine peel polyphenol hybrid liposomes were prepared according to the method of Example 1, except that the tangerine peel polyphenol hybrid liposomes of this comparative example did not contain cell penetrating factors and were prepared from the following components: sunflower seed lecithin, cholesterol and tangerine peel polyphenol extract in a weight ratio of 10:2:3.

[0088] The particle size of the tangerine peel polyphenol hybrid liposomes prepared in this comparative example is 85.6±0.45nm.

[0089] Test Example 1

[0090] The cellular uptake of the citrus polyphenol hybrid liposomes of Examples 1 to 5 and Comparative Example 1 was evaluated, and the specific method was as follows: during the preparation of the citrus polyphenol hybrid liposomes, the fluorescent dye coumarin 6 (a polyphenol fluorescent dye) was added to label the liposomes, RAW264.7 cells were inoculated in a 6-well plate (6×105 cells / well), cultured for 24 hours, and then treated with 2 μg / mL fluorescent labeled liposomes for 2 hours. The cells were collected and made into a single cell suspension, and the fluorescence intensity was analyzed by flow cytometry (FACSCalibur, BD, USA). The test results are listed in Table 1.

[0091] The cells were treated with free fluorescent polyphenol dye and the fluorescently labeled liposomes of Example 3 for 2 h, and the cell nuclei of the cells were stained with DAPI dye and the cell uptake was observed under an inverted fluorescence microscope. Figure 4 As shown, after the free fluorescent polyphenols were incubated with RAW264.7 cells for 2 hours, only a small amount of fluorescence was detected in the cells, because the free fluorescent polyphenols were difficult to enter the cells in the form of passive diffusion; while after the fluorescent polyphenol-labeled tangerine red polyphenol hybrid liposomes were incubated with RAW264.7 cells for 2 hours, strong fluorescence was detected in the cells, because the cell penetrating factors on the surface of the hybrid liposomes can interact with the cell membrane to enhance the ability to penetrate the cell membrane, thereby increasing the amount of substances carried into the cells.

[0092] Table 1

[0093] Mean fluorescence intensity Example 1 3291±85 Example 2 3076±85 Example 3 3233±72 Example 4 2306±88 Example 5 2754±87 Comparative Example 1 2280±42

[0094] Test Example 2

[0095] The lipopolysaccharide (LPS)-induced anti-inflammatory effect of the citrus polyphenol hybrid liposomes of Examples 1 to 5 and Comparative Example 1 was evaluated. The specific method was as follows: RAW264.7 cells in the logarithmic growth phase were adjusted to a concentration of 5×10 5 / mL, 100 μL per well was inoculated in a 96-well plate, and placed in a 37°C, 5% CO2 incubator for 24 hours until the cells adhered to the wall. A blank group (cells), a model group (cells + LPS), a control group (cells + tangerine red polyphenol extract + LPS) and an experimental group (cells + liposomes + LPS) were set up, wherein the control group added tangerine red polyphenol extract at a final concentration of 0.2 mg / mL, and the experimental group added tangerine red polyphenol hybrid liposomes of Examples 1 to 5 and Comparative Example 1, and the final concentration of tangerine red polyphenol extract was 0.2 mg / mL to pretreat RAW264.7 cells for 2 hours. Then, except for the blank group, LPS (final concentration 1 μg / mL) was added to the rest to induce inflammation in RAW264.7. The Griess method was used to determine the changes in the secretion of nitric oxide (NO) of RAW264.7 cells, and ELISA was used to detect the changes in the secretion of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and monocyte chemoattractant protein-1 (MCP-1) of RAW264.7 cells. The results are listed in Table 2.

[0096] Table 2

[0097]

[0098] As can be seen from Table 2, the tangerine peel polyphenol hybrid liposomes of Examples 1 to 5 are beneficial to reducing the secretion of NO, IL-6 and MCP-1 of RAW264.7 cells after LPS stimulation, showing obvious anti-inflammatory ability, among which the tangerine peel polyphenol hybrid liposomes of Examples 1 to 3 have better effects.

[0099] Test Example 3

[0100] The citrus red polyphenol hybrid liposomes of Examples 1 to 5 and Comparative Example 1 were evaluated for their anti-inflammatory effects induced by ultraviolet B (UVB). The specific method was as follows: HaCaT skin cells were adjusted to a concentration of 5×10 5 / mL, 100 μL per well was inoculated in a 96-well plate and placed in a 37°C, 5% CO2 incubator for 24 hours until the cells adhered to the wall. A blank group (cells), a model group (cells + UVB), a control group (cells + tangerine peel polyphenol extract + UVB) and an experimental group (cells + liposomes + UVB) were set up, wherein the control group added tangerine peel polyphenol extract at a final concentration of 0.2 mg / mL, and the experimental group added tangerine peel polyphenol hybrid liposomes of Examples 1 to 5 and Comparative Example 1, and the final concentration of tangerine peel polyphenol extract was 0.2 mg / mL to pretreat HaCaT cells for 2 hours. Then, except for the blank group, the rest were irradiated with a UVB dose of 40 mJ / cm 2 The treatment induced inflammation in HaCaT cells. The secretion of tumor necrosis factor-α (TNF-α) and interleukin-8 (IL-8) in HaCaT cells was detected by ELISA, and the results are listed in Table 3.

[0101] Table 3

[0102] TNF-α secretion, pg / mL IL-8 secretion, pg / mL Example 1 30.22±0.39 165.00±3.80 Example 2 30.78±0.41 168.21±3.12 Example 3 29.87±0.52 162.95±2.90 Example 4 33.97±0.29 174.70±2.28 Example 5 32.53±0.66 173.48±4.16 Comparative Example 1 37.72±0.39 178.64±2.73 Control group 56.89±0.89 197.9±5.03 Model Group 76.06±0.39 235.21±2.33 Blank Group 11.89±0.79 58.06±0.99

[0103] As can be seen from Table 3, the tangerine peel polyphenol hybrid liposomes of Examples 1 to 5 are beneficial to reducing the secretion of TNF-α and IL-8 of skin cells HaCaT caused by UVB radiation, and have the effect of improving skin cell inflammation, among which the tangerine peel polyphenol hybrid liposomes of Examples 1 to 3 have better effects.

[0104] Examples 6 to 12 are used to illustrate the lotions of the present disclosure.

[0105] Example 6

[0106] The tangerine peel polyphenol hybrid liposome of Example 1 was used to prepare a lotion. The lotion of this example was prepared from the following components: 5.6% by weight of tangerine peel polyphenol hybrid liposome, 0.32% by weight of β-glucan, 0.8% by weight of sodium pyrrolidonecarboxylate, 0.08% by weight of potassium cinnamate, and the balance of water. The preparation method is as follows:

[0107] Add the thickener to sterile water (the weight ratio of the thickener to sterile water is 1:37) according to the ratio and stir evenly to completely dissolve the thickener to obtain solution 1; add the tangerine peel polyphenol hybrid liposome to sterile water (the weight ratio of the tangerine peel polyphenol hybrid liposome to sterile water is 1:14) according to the ratio and ultrasonically dissolve and disperse to obtain solution 2; place solution 1 and solution 2 in a vacuum mixer, add moisturizer and antibacterial agent to the solution according to the ratio, seal the system, set the vacuum degree: -0.05MPa, the stirring speed: 3000rpm, the stirring time: 30min, and vacuum stir and mix evenly to obtain a lotion product.

[0108] Example 7

[0109] The tangerine peel polyphenol hybrid liposome of Example 2 was used to prepare a lotion. The lotion of this example was prepared from the following components: 5.0 wt% of tangerine peel polyphenol hybrid liposome, 0.1 wt% of flaxseed gum, 0.35 wt% of hyaluronic acid, 0.35 wt% of epigallocatechin gallate, and the balance of water. The preparation method is as follows:

[0110] Add the thickener to sterile water (the weight ratio of the thickener to sterile water is 1:80) according to the ratio and stir evenly to completely dissolve the thickener to obtain solution 1; add the tangerine peel polyphenol hybrid liposome to sterile water (the weight ratio of the tangerine peel polyphenol hybrid liposome to sterile water is 1:11) according to the ratio and ultrasonically dissolve and disperse to obtain solution 2; place solution 1 and solution 2 in a vacuum mixer, add a moisturizer and an antibacterial agent to the solution according to the ratio, seal the system, set the vacuum degree to 0.02 MPa, the stirring speed to 6000 rpm, the stirring time to 10 min, and vacuum stir and mix evenly to obtain a lotion product.

[0111] Example 8

[0112] The tangerine peel polyphenol hybrid liposome of Example 3 was used to prepare a lotion. The lotion of this example was prepared from the following components: 4.7 wt % of tangerine peel polyphenol hybrid liposome, 0.24 wt % of Tremella polysaccharide, 1.25 wt % of L-serine, 0.26 wt % of tannic acid and the balance of water. The preparation method is as follows:

[0113] Add the thickener to sterile water (the weight ratio of the thickener to sterile water is 1:25) according to the ratio and stir evenly to completely dissolve the thickener to obtain solution 1; add the tangerine peel polyphenol hybrid liposome to sterile water (the weight ratio of the tangerine peel polyphenol hybrid liposome to sterile water is 1:31) according to the ratio and ultrasonically dissolve and disperse to obtain solution 2; place solution 1 and solution 2 in a vacuum mixer, add moisturizer and antibacterial agent to the solution according to the ratio, seal the system, set the vacuum degree to 0.09 MPa, the stirring speed to 1500 rpm, the stirring time to 60 min, and obtain a lotion product after vacuum stirring and mixing evenly.

[0114] Example 9

[0115] The tangerine peel polyphenol hybrid liposomes of Example 4 were used to prepare lotion according to the method of Example 6.

[0116] Example 10

[0117] The tangerine peel polyphenol hybrid liposomes of Example 5 were used to prepare lotion according to the method of Example 6.

[0118] Embodiment 11

[0119] A lotion was prepared according to the method of Example 6. The lotion of this example was prepared from the following components: 2.8 wt % of tangerine peel polyphenol hybrid liposome, 0.4 wt % of β-glucan, 2.3 wt % of sodium pyrrolidonecarboxylate, 0.75 wt % of tannic acid and the remainder of water.

[0120] Example 12

[0121] A lotion was prepared according to the method of Example 6. The lotion of this example was prepared from the following components: 8.2 wt% of tangerine peel polyphenol hybrid liposomes, 0.01 wt% of β-glucan, 0.12 wt% of sodium pyrrolidonecarboxylate, 0.01 wt% of potassium cinnamate and the remainder of water.

[0122] Comparative Example 2

[0123] The tangerine peel polyphenol hybrid liposomes of Comparative Example 1 were used to prepare lotion according to the method of Example 6.

[0124] Test Example 4

[0125] The moisturizing and anti-inflammatory effects of the lotions of Examples 6 to 12 and Comparative Example 2 were evaluated, and the specific method was as follows: SKH-1 hairless mice were selected for the moisturizing effect evaluation, and a blank group (hairless mice), a control group (hairless mice + water) and an experimental group (hairless mice + lotion) were set up, with 8 hairless mice in each group. The initial skin moisture content in the specified area on the back of the hairless mice was first measured, wherein the control group applied pure water on the specified backpack area of ​​the hairless mice, and the experimental group applied lotion on the specified backpack area of ​​the hairless mice. After 30 minutes, the moisture content of the hairless mouse skin was measured to determine the moisturizing effect.

[0126] Evaluation of the anti-inflammatory effect SKH-1 hairless mice were selected, and blank group (hairless mice), control group (hairless mice + TPA treatment) and experimental group (hairless mice + TPA treatment + lotion) were set up, with 8 hairless mice in each group, and TPA (phorbol 12-myristate 13-acetate dissolved in acetone, 3μg / 20μL) was used to stimulate the back skin to induce skin inflammation. The back of the blank group was smeared with acetone, the back of the control group was stimulated with TPA, and the back of the experimental group was stimulated with TPA for 3h and then coated with Examples 6 to 12 and Comparative Example 2 lotion. After repeating this process for five consecutive days, the back tissue of the mice was taken and the IL-8 secretion in the back tissue of the mice was detected by ELISA. The results are listed in Table 4.

[0127] Table 4

[0128] Skin moisture content, % IL-8 secretion, pg / mL Example 6 48.7±1.6 21.38±1.54 Example 7 49.2±2.7 18.85±2.90 Example 8 45.3±2.1 22.61±3.21 Example 9 42.5±1.2 24.83±2.24 Example 10 41.2±1.9 29.20±3.37 Embodiment 11 39.2±1.5 25.96±4.21 Example 12 40.8±2.5 30.79±5.22 Comparative Example 2 37.3±2.2 32.76±2.59 Control group 33.1±1.7 34.78±5.49 Blank Group 32.5±2.8 11.21±2.1

[0129] As can be seen from Table 4, the lotions prepared in Examples 6 to 12 have improved moisturizing and anti-inflammatory effects, among which the lotions in Examples 6 to 8 have better effects.

[0130] The preferred embodiments of the present disclosure are described in detail above in conjunction with the accompanying drawings; however, the present disclosure is not limited to the specific details in the above embodiments. Within the technical concept of the present disclosure, a variety of simple modifications can be made to the technical solution of the present disclosure, and these simple modifications all fall within the protection scope of the present disclosure.

[0131] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present disclosure will not further describe various possible combinations.

[0132] In addition, various embodiments of the present disclosure may be arbitrarily combined, and as long as they do not violate the concept of the present disclosure, they should also be regarded as the contents disclosed by the present disclosure.

Claims

1. A tangerine peel polyphenol hybrid liposome, characterized in that: The tangerine peel polyphenol hybrid liposome comprises phospholipids, cholesterol, cell penetrating factors and tangerine peel polyphenol extract; the weight ratio of the phospholipids, the cholesterol, the cell penetrating factors and the tangerine peel polyphenol extract is 10: (0.5-3): (0.5-8): (0.1-6).

2. The tangerine peel polyphenol hybrid liposome according to claim 1, wherein: The phospholipids include one or more of sunflower seed phospholipids, soybean lecithin and sphingomyelin.

3. The tangerine peel polyphenol hybrid liposome according to claim 1, wherein: The cell penetrating factor comprises one or more of phospholipid-polyethylene glycol-octaarginine, phospholipid-polyethylene glycol-nonaarginine and phospholipid-polyethylene glycol-cell penetrating peptide.

4. A method for preparing the tangerine peel polyphenol hybrid liposome according to any one of claims 1 to 3, characterized in that: The method includes: The tangerine peel polyphenol extract, phospholipids, cholesterol, cell penetrating factor and solvent are stirred and mixed to obtain a mixed solution; The mixed solution is subjected to reduced pressure rotary evaporation to obtain a lipid film; The lipid film is mixed with the first sterile water, and then subjected to ultrasonic dispersion treatment, and then freeze-dried to obtain the tangerine peel polyphenol hybrid liposome.

5. The method according to claim 4, wherein: The solvent is ethanol and chloroform, and the weight ratio of the phospholipid, the ethanol and the chloroform is 1:(14-34):(7-17); and / or, The stirring and mixing conditions include: a stirring speed of 100 to 500 rpm.

6. The method according to claim 4, wherein: The conditions of the reduced pressure rotary evaporation include: vacuum degree of 0.01-0.10 MPa, temperature of 45-60° C., and rotation speed of 50-200 rpm; and / or, The conditions of the ultrasonic dispersion treatment include: ultrasonic power of 400-800W and time of 3-8min.

7. A lotion, characterized in that: The lotion comprises the tangerine peel polyphenol hybrid liposome as described in any one of claims 1 to 3; based on the total weight of the lotion, the lotion comprises 2.8 to 8.2 weight % of the tangerine peel polyphenol hybrid liposome, 0.01 to 0.4 weight % of a thickener, 0.12 to 2.3 weight % of a moisturizer, 0.01 to 0.75 weight % of an antibacterial agent and the remainder water.

8. The lotion according to claim 7, wherein: The thickener comprises one or more of β-glucan, flaxseed gum and tremella polysaccharide; and / or, The moisturizing agent includes one or more of sodium pyrrolidone carboxylate, potassium pyrrolidone carboxylate, L-serine, hyaluronic acid and ceramide; and / or, The antibacterial agent includes one or more of potassium cinnamate, epigallocatechin gallate and tannic acid.

9. A method for preparing the lotion according to claim 7 or 8, characterized in that: The method includes: mixing the thickener with the second sterile water to obtain a thickener solution; Mixing the tangerine peel polyphenol hybrid liposome with the third sterile water to obtain a tangerine peel polyphenol hybrid liposome solution; The thickener solution, the citrus aurant polyphenol hybrid liposome solution, the moisturizer and the antibacterial agent are mixed and then vacuum stirred to obtain a lotion.

10. The method according to claim 9, wherein: The vacuum stirring conditions include: vacuum degree of -0.05 MPa to 0.09 MPa, stirring speed of 1500 to 6000 rpm, and stirring time of 10 to 60 min.

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