Antioxidant and anti-aging red bean and bilberry liposome gel capable of rapidly repairing skin collagen fibers damaged by specific dermatitis and preparation method of antioxidant and anti-aging red bean and bilberry liposome gel

Abstract

The invention discloses antioxidant and anti-aging red bean and bilberry liposome gel capable of rapidly repairing collagen fibers of skin damaged by specific dermatitis and a preparation method of the antioxidant and anti-aging red bean and bilberry liposome gel, relates to the field of medicines, and aims to solve the problem of lack of natural antioxidant, anti-aging and anti-sensitive skin liposome gel in the prior art. The preparation method of the red bean and bilberry liposome gel comprises the following steps: step 1, preparing a red bean and bilberry alcohol extract; 2, enriching and purifying the red bean and bilberry polyphenol to obtain a red bean and bilberry polyphenol extracting solution; step 3, performing freeze drying treatment to obtain red bean and cowberry freeze-dried powder; step 4, (1) dissolving soybean lecithin and cholesterol in chloroform, and evaporating to prepare a film to obtain a lipoid film; (2) adding freeze-dried red bean and cowberry powder and a surfactant into water, uniformly mixing the freeze-dried red bean and cowberry powder and the surfactant with the lipoid film, and performing rotary evaporation and shaking to prepare a red bean and cowberry liposome suspension; (3) carrying out ultrasonic treatment, filtering and granulating to obtain the red bean and bilberry lipidosome; and step 5, adding the gel into the red bean and bilberry liposome, uniformly mixing, and standing to obtain the red bean and bilberry liposome gel.

Description

Technical Field

[0001] The invention relates to the field of medical technology, in particular to an anti-oxidative and anti-aging red bean and bilberry liposome gel for rapidly repairing skin collagen fibers damaged by atopic dermatitis and a preparation method thereof. Background Art

[0002] Free radical oxidation and skin inflammation have been bothering most people, especially women between 30 and 50 years old. Regarding aging, scientists believe that one of the important reasons is the gradual oxidation of the human body. The so-called "oxidation" is like iron rusting and peeled apples changing color. The human body is also experiencing the "rusting" oxidation process every moment. Therefore, enhancing the body's antioxidant capacity is the top priority for anti-aging. Therefore, safe, natural and additive-free products are urgently called for and needed by people.

[0003] In pursuit of effective results, most people use cosmetics containing antibiotics and hormone drugs, which will damage the skin's microecology, cause the stratum corneum to become thinner, make the skin sensitive, etc., and make skin problems more serious.

[0004] In recent years, liposomes have attracted wide attention due to their good biocompatibility, no irritation to the skin, enhanced skin closure and hydration, reduced water loss, enhanced moisturizing properties, small particle size and rapid film formation, and are widely used in the pharmaceutical and cosmetic industries. However, the existing technology lacks natural liposome gels with antioxidant, anti-aging and anti-sensitive skin properties. Summary of the invention

[0005] The technical problems to be solved by the present invention are:

[0006] The prior art lacks natural anti-oxidation, anti-aging, and anti-sensitive skin liposome gel.

[0007] The present invention adopts the following technical solutions to solve the above technical problems:

[0008] The invention provides an anti-oxidation and anti-aging red bean and bilberry liposome gel for quickly repairing skin collagen fibers damaged by specific dermatitis. The gel comprises red bean and bilberry liposomes and gel. Every 100 ml of the red bean and bilberry liposomes are dispersed in 15.5-133 g of gel.

[0009] Furthermore, the raw materials of the red bean and cranberry liposomes include red bean and cranberry polyphenols, soybean lecithin, cholesterol and a surfactant, and the soybean lecithin and cholesterol form a lipid film that is coated on the surface of the red bean and cranberry polyphenols.

[0010] Furthermore, the mass ratio of soybean lecithin to cholesterol in the lipid film is 10:3, and Tween 80 is used as the surfactant.

[0011] Furthermore, the gel comprises 0.5 to 5 parts of poloxamer 188, 12 to 125 parts of poloxamer 407 and 3 parts of propylene glycol in parts by mass.

[0012] The present invention provides a method for preparing an anti-oxidative and anti-aging red bean and bilberry liposome gel for rapidly repairing skin collagen fibers damaged by atopic dermatitis, comprising the following steps:

[0013] Step 1, washing fresh blueberry, adding into ethanol solution for ultrasonic extraction, and obtaining blueberry ethanol extract;

[0014] Step 2, enriching and purifying the bilberry polyphenols in the bilberry alcohol extract to obtain a bilberry polyphenol extract;

[0015] Step 3, freeze-drying the blueberry polyphenol extract to obtain blueberry freeze-dried powder;

[0016] Step 4: ① dissolve soybean lecithin and cholesterol in chloroform according to a certain proportion, and then evaporate to obtain a lipid film; ② add the freeze-dried red bean and blueberry powder and a surfactant into water and mix them with the lipid film, and then perform rotary evaporation and shaking at 30-40° C. to obtain a red bean and blueberry liposome suspension; ③ ultrasonically treat the red bean and blueberry liposome suspension, filter, and granulate to obtain red bean and blueberry liposome;

[0017] Step 5: Evenly mix poloxamer 188, poloxamer 407 and propylene glycol according to their weight proportions to obtain a gel, add the gel to the red bean and bilberry liposomes, mix well, and let stand to obtain the red bean and bilberry liposome gel.

[0018] Furthermore, the concentration of the ethanol solution in step 1 is 75%, the heating time is 25 min to 45 min under the condition of ultrasonic power of 600 W to 800 W, the temperature is 55° C., and the material-liquid ratio is 1:3-1:4.

[0019] Furthermore, in step 2, YWD07 macroporous resin is used to enrich and purify red bean bilberry polyphenols, specifically: wet column packing, distilled water compacting the adsorption column, static adsorption for 12h-18h, then washing the column with distilled water for chromatography, removing sugars and small molecules in the crude extract, and finally eluting with 80%-90% ethanol to obtain red bean bilberry polyphenol extract.

[0020] Furthermore, the freeze-drying time in step three is 24h-36h.

[0021] Furthermore, in step five, 15.5 to 133 g of gel is added to every 100 ml of red bean and blueberry liposomes.

[0022] Furthermore, the standing time in step 5 is not less than 24 hours.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] 1. The nano-drug delivery system is used to encapsulate the effective ingredients of traditional Chinese medicine to produce nano-preparations of traditional Chinese medicine, so that blueberry polyphenols can be quickly introduced into the skin, increasing the bioavailability, stability and solubility of the drug and reducing irritation to the skin.

[0025] 2. The liposome prepared by the present invention has an encapsulation rate of 91.01% measured by using the blueberry polyphenol content as an indicator, breaking the limitation of traditional encapsulation of a single substance and solving the problem of low encapsulation rate caused by traditional liposome encapsulation of multiple components. It is a breakthrough in the field of liposome preparation.

[0026] 3. The liposomes prepared by the present invention have a strong antibacterial effect and have a certain inhibitory effect on hemolytic streptococci, Escherichia coli, Staphylococcus aureus, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is a transmission electron microscope image of the liposome gel in an embodiment of the present invention;

[0028] Figure 2 is a transmission electron microscope image of the liposome in an embodiment of the present invention;

[0029] Figure 3 The antagonistic graphs of the liposomes in the embodiments of the present invention against hemolytic streptococci, Escherichia coli, and Staphylococcus aureus respectively;

[0030] Figure 4 This is a diagram showing the regulatory effect of liposomes on skin flora in an embodiment of the present invention;

[0031] Figure 5 ABTS for the in vitro antioxidant test in the embodiment of the present invention + Clearance plot;

[0032] Figure 6 This is a graph of Dpph free radical scavenging rate in an in vitro antioxidant test in an embodiment of the present invention;

[0033] Figure 7 This is a total reducing capacity diagram of the in vitro antioxidant test in the embodiment of the present invention;

[0034] Figure 8 is the response surface of the membrane material ratio, hydration temperature and drug dosage to the encapsulation efficiency in the embodiment of the present invention;

[0035] Fig. 9 It is a microscopic image of a skin tissue section in an embodiment of the present invention. DETAILED DESCRIPTION

[0036] In order to enable those skilled in the art to better understand the scheme of the present invention, exemplary implementations or embodiments of the present invention will be described below in conjunction with the accompanying drawings. Obviously, the described implementations or embodiments are only implementations or embodiments of a part of the present invention, not all of them. Based on the implementations or embodiments of the present invention, all other implementations or embodiments obtained by ordinary technicians in the field without creative work should fall within the scope of protection of the present invention.

[0037] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, specific embodiments of the present invention are described in detail below with reference to the accompanying drawings.

[0038] The present invention provides a method for preparing an anti-oxidative and anti-aging red bean and bilberry liposome gel for rapidly repairing skin collagen fibers damaged by atopic dermatitis, comprising the following steps:

[0039] Step 1: wash fresh cranberry, add it to 75% ethanol solution, the solid-liquid ratio is 1:3-1:4, homogenize, ultrasonicate at 600W-800W, heat at 55°C for 25min-45min, to obtain cranberry alcohol extract; the concentration of the cranberry alcohol extract is 18g / 100mL-20g / 100mL.

[0040] Step 2: YWD07 macroporous resin is used to enrich and purify the red bean bilberry polyphenols, specifically: wet column packing, distilled water compacting adsorption column, static adsorption for 12h-18h, and then washing the column chromatography with distilled water at a flow rate of 2mL / min to remove sugars and small molecules in the crude extract. After about two column volumes, Fehling's reagent (Fehling's reagent A: 0.1g / mLNaOH, Fehling's reagent B: 0.05g / mL CuSO4) is used to detect whether the effluent contains residual sugar. Finally, 80%-90% ethanol is used for elution at an elution flow rate of 2mL / min to obtain a red bean bilberry polyphenol extract.

[0041] Step 3: freeze-dry the blueberry polyphenol extract for 24 hours to 36 hours to obtain loose and porous blueberry freeze-dried powder.

[0042] Step 4: ① Dissolve soybean lecithin and cholesterol in chloroform at a mass ratio of 10:3, and then use a rotary evaporator to evaporate and form a film at 32°C and 60rpm to obtain a lipid film; ② Add 10-30mg of red bean and blueberry freeze-dried powder and 20ul of Tween 80 into water and mix them with the lipid film, and then shake them by rotary evaporation at 30°C-40°C under vacuum to obtain a red bean and blueberry liposome suspension; ③ Use probe ultrasound to sonicate the red bean and blueberry liposome suspension, filter it, and granulate it to obtain red bean and blueberry liposomes.

[0043] Step 5, preparation of liposome gel: weigh 0.5-5 g of poloxamer 188, 12-125 g of poloxamer 407 and 3 ml of propylene glycol and mix them evenly to obtain a gel, add 15.5-133 g of the gel to every 100 ml of red bean and cranberry liposomes, mix well, and let stand at 4°C for not less than 24 hours to obtain the red bean and cranberry liposome gel.

[0044] The single factor investigation on bilberry extraction process was carried out, and the results are shown in Tables 1-5.

[0045] Table 1

[0046] Alcohol concentration Content (mg / g) 70％ 2.727484721 75％ 2.794624179 80％ 2.751522345 85％ 2.678459258 90％ 2.641819085

[0047] Table 2

[0048] Ultrasonic power (w) Content (mg / g) 500 2.188338565 600 2.687998358 700 3.11795925 800 2.814436549

[0049] Table 3

[0050] Material-liquid ratio Content (mg / g) 1:3 2.535690101 1:4 2.792207423 1:5 2.264933094 1:6 1.252730047 1:7 0.597348394

[0051] Table 4

[0052] Temperature(℃) Content (mg / g) 25 3.235623119 35 3.340990469 45 3.50137309 55 3.678725243 65 3.509887996

[0053] Table 5

[0054]

[0055]

[0056] The optimal ranges of various process parameters are shown in Tables 6 and 7.

[0057] Table 6

[0058]

[0059] Table 7

[0060]

[0061] The total amount of bilberry polyphenols and the free bilberry polyphenols content in red bean bilberry liposomes were determined by dialysis method:

[0062] Determination of total drug dosage: Select a dialysis bag with specification of MD34 (8000-14000), take 20 mg of red bean bilberry powder, add it to the dialysis bag of MD34 (8000-14000), and then place it in 250 ml of ultrapure water (37°C, 400r / min stirring). After dialysis equilibrium after 12 hours, use a rotary evaporator to concentrate to 10 ml, absorb 2 ml, and use ultraviolet to detect the total drug dosage W of bilberry polyphenols. 总 .

[0063] Determination of free drugs: Use a dialysis bag with a specification of MD34 (8000-14000), take 10ml of red bean bilberry liposome suspension, add it to the dialysis bag of MD34 (8000-14000), and then place it in 250ml ultrapure water (37℃, 400r / min stirring). After dialysis equilibrium for 12h, use a rotary evaporator to concentrate to 10ml, take 2ml, and use ultraviolet to detect the amount of free bilberry polyphenol drugs W 游 .

[0064] Determination of encapsulation efficiency and drug loading:

[0065] Encapsulation efficiency is an important indicator for evaluating liposomes in formulation and process screening, optimization and quality evaluation.

[0066] Encapsulation efficiency calculation formula: Encapsulation efficiency (%) = (W 总 -W 游 ) / W 总 ×100%

[0067] The drug loading calculation formula is: drug loading (%) = (drug dosage - free drug amount) / (liposome dosage + drug dosage - free drug amount) × 100%.

[0068] Liposome single factor investigation

[0069] Effect of total lipid amount on cranberry liposomes: the ratio of cholesterol to soybean lecithin was 3:10, cranberry 20.00 mg, hydration liquid volume 20 mL, hydration temperature 36°C, and hydration time 45 min. The effect of total lipid amount (208 mg, 260 mg, 312 mg, 364 mg, 416 mg) on ​​the encapsulation efficiency of cranberry liposomes was investigated.

[0070] Effect of the ratio of cholesterol to soybean lecithin on cranberry liposomes: cranberry 20.00 mg, hydration liquid volume 20 mL, hydration temperature 36°C, hydration time 45 min. The effect of the ratio of cholesterol to soybean lecithin (1:12, 2:11, 3:10, 4:9) on the encapsulation efficiency of cranberry liposomes was investigated.

[0071] Effect of hydration medium on cranberry liposomes: cranberry 20.00 mg, hydration liquid volume 20 mL, hydration temperature 36°C, hydration time 45 min, to investigate the effect of hydration medium (phosphate buffer pH (5.6, 6, 6.4, 6.8, 7.2) and water) on the encapsulation efficiency of cranberry liposomes.

[0072] Effect of hydration temperature on cranberry liposomes: 20.00 mg of cranberry, hydration liquid volume of 20 mL, hydration time of 45 min, and the effect of hydration temperature (30°C, 32°C, 36°C, 40°C, 44°C) on the encapsulation efficiency of cranberry liposomes were investigated.

[0073] Effect of dosage of blueberry freeze-dried powder on blueberry liposomes: hydration liquid volume 20mL, hydration time 45min, investigate the effect of dosage (10mg, 15mg, 20mg, 25mg, 30mg) on ​​the encapsulation efficiency of blueberry liposomes.

[0074] Table 8

[0075] Total lipids mg Encapsulation efficiency 208 79.91％ 260 82.98％ 312 83.34％ 364 77.88％ 416 77.69％

[0076] Table 9

[0077]

[0078]

[0079] Table 10

[0080] PH Encapsulation efficiency 5.6 83.81％ 6 83.94％ 6.4 85.27％ 6.8 83.78％ 7.2 83.22％ water 90％

[0081] Table 11

[0082] Hydration temperature (℃) Encapsulation efficiency 30 86.70％ 32 91.10％ 36 89.00％ 40 88.70％ 44 87.80％

[0083] Table 12

[0084] Dosage of Red Bean and Blueberry Freeze-dried Powder (mg) Encapsulation efficiency 10 87.01％ 15 89.78％ 20 91.10％ 25 90.72％ 30 89.43％

[0085] like Figure 8 As shown, response surface optimization was performed and the results are shown in Table 13.

[0086] Table 13

[0087]

[0088] The beneficial effects of the present invention are described in detail by the following examples.

[0089] Example 1

[0090] The present invention provides a method for preparing an anti-oxidative and anti-aging red bean and bilberry liposome gel for rapidly repairing skin collagen fibers damaged by atopic dermatitis, comprising the following steps:

[0091] Step 1: Wash fresh blueberry, add it to 75% ethanol solution, with a solid-liquid ratio of 1:4, homogenize, ultrasonicate at 800W, and heat at 55°C for 30min to obtain blueberry alcohol extract;

[0092] Step 2: YWD07 macroporous resin was used to enrich and purify the red bean bilberry polyphenols, specifically: wet column packing, distilled water compacted adsorption column, static adsorption for 15 hours, and then the column was washed with distilled water at a flow rate of 2mL / min to remove sugar and small molecules in the crude extract. After about two column volumes, Fehling's reagent (Fehling's reagent A: 0.1g / mL NaOH, Fehling's reagent B: 0.05g / mL CuSO4) was used to detect whether the effluent contained residual sugar. Finally, 80% ethanol was used for elution at a flow rate of 2mL / min to obtain the red bean bilberry polyphenol extract.

[0093] Step 3: freeze-dry the blueberry polyphenol extract for 36 hours to obtain loose and porous blueberry freeze-dried powder.

[0094] Step 4: ① Dissolve soybean lecithin and cholesterol in chloroform at a mass ratio of 10:3, and then use a rotary evaporator to evaporate and form a film at 32°C and 60rpm to obtain a lipid film; ② Add 20 mg of red bean and blueberry freeze-dried powder and 20 ul of Tween 80 into water and mix them with the lipid film, and then shake them by rotary evaporation at 35°C under vacuum to obtain a red bean and blueberry liposome suspension; ③ Use probe ultrasound to sonicate the red bean and blueberry liposome suspension, filter it, and granulate it to obtain red bean and blueberry liposomes.

[0095] Step 5, preparation of liposome gel: weigh 3 g of poloxamer 188, 70 g of poloxamer 407 and 3 ml of propylene glycol and mix them evenly to obtain a gel, dissolve the gel in 100 ml of red bean and bilberry liposomes, mix well, and let stand at 4° C. for 30 h to obtain the red bean and bilberry liposome gel.

[0096] Appearance of liposomes:

[0097] Take an appropriate amount of the red bean and cranberry liposomes of Example 1 and dilute it with water to about 10 times the volume. Then take 20 μL of the dilution and place it on a copper grid to absorb for five minutes. Then add an appropriate amount of 1% phosphotungstic acid to stain for 1 minute. After natural air drying, observe its shape under a transmission electron microscope (TEM) and take a picture. Figure 1 As shown, the liposomes are spherical, have complete structures, and are evenly dispersed.

[0098] Take an appropriate amount of the red bean and cranberry liposome suspension of Example 1, dilute it with 10 times the volume of the aqueous solution, and analyze the particle size distribution and Zeta potential of the liposome using a laser particle size analyzer. The particle size distribution is relatively uniform, as shown in FIG. Figure 2The particle size is about 136 nm, the polydispersity index (PDI) is 0.207, the Zeta potential is -48.56 mV, and the solution is stable. Since the liposome particle size reaches the nanometer level of 136 nm, the mixture of blueberry polyphenols is quickly introduced into the skin, enhancing the antioxidant activity of polyphenols on the skin. The liposome gel prepared by the present invention has an effect on the antioxidant activity of mouse skin.

[0099] Test of influencing factors: Placed in an environment of high temperature, high humidity and strong light (60°C, RH90±5%, 4500Lx±500Lx) for 5d and 10d, the results are shown in Table 14 and Table 15 respectively. There is no obvious change in particle size and no precipitation.

[0100] Table 14

[0101] high temperature High humidity Strong light exposure normal Particle size 111.48 114.23 113.13 113.13 PDI 0.208 0.224 0.212 0.212 zate potential -18.22v -22.45v -32.79v -25.37v

[0102] Table 15

[0103] high temperature High humidity Strong light exposure normal Particle size 147.77 155.33 131.72 115.94 PDI 0.254 0.225 0.207 0.225 zate potential -21.98v -24.32v -27.82v -27.82v

[0104] Stability test: The prepared liposomes were placed in a sample bottle for 28 days. The appearance, particle size, zate potential and PDI of the liposomes were observed and measured on the 7th, 14th, 21st and 28th days, respectively. The results are shown in Table 16.

[0105] Table 16

[0106]

[0107] Example 2

[0108] The red bean bilberry crude extract of embodiment 1 is diluted to the series concentration of 2,1,0.5,0.25,0.125,0.065g / mL with deionized water.1mL bacteria (hemolytic streptococcus, Escherichia coli, Staphylococcus epidermidis, Staphylococcus aureus and Salmonella) liquid is joined in the culture dish that culture medium is housed, with spreading rod, bacteria liquid is evenly coated on culture medium, get Oxford cup and be placed on culture medium surface and add 100 μ L different extracts respectively in Oxford cup, use cefixime as positive control.After culture dish is put into constant temperature incubator and cultured at constant temperature for 24h, take out and measure the inhibition zone diameter, average.Each concentration is done 2 parallel samples, and the minimum concentration that bacterium colony is suppressed completely is minimum inhibitory concentration.

[0109] like Figure 3 As shown, hemolytic streptococci: the minimum inhibitory concentration is 0.5g / ml, the diameter of the inhibition zone is 1.5cm ( Figure 3 (a)); the minimum inhibitory concentration of Staphylococcus epidermidis is 1g / ml and the diameter of the inhibition zone is 1.8cm ( Figure 3(b) The minimum inhibitory concentration of Staphylococcus aureus is 1g / ml and the diameter of the inhibition zone is 1.6cm Figure 3 (c)), Escherichia coli: minimum inhibitory concentration of 1g / ml, inhibition zone diameter: 1.4cm, Salmonella minimum inhibitory concentration of 1g / ml, inhibition diameter 1.5cm. It can be seen that the red bean bilberry crude extract of the present invention has a certain inhibitory effect on hemolytic streptococci, Escherichia coli, Staphylococcus epidermidis, Staphylococcus aureus and Salmonella.

[0110] Example 2

[0111] 80 mice were randomly divided into blank control group, model group, mupirocin (1.67 mg / kg) group, low, medium and high dose groups of red bean and bilberry liposome gel (0.5 ml, 1 ml and 2 ml of red bean and bilberry liposome gel were applied to the back of mice, respectively), VE group and gel matrix group, 10 mice in each group. The hair on the back of mice (2 cmx3 cm) was removed 1 day before the experiment. On the 1st and 3rd days of the experiment, except for the blank control group, 100 μL and 50 μL of 1% DNCB were applied to all the hair removal areas on the back of mice in the other groups, respectively. From the 7th day, 0.5% DNCB was applied every other day; the control group was applied with the corresponding matrix solution (acetone-olive oil = 3:1), and the model was considered to be successful when desquamation, erythema, edema and scab appeared on the back of mice. 8 hours after each application of 0.5% DNCB, the red bean and bilberry liposome gel groups, the gel matrix group (gel without bilberry liposomes prepared according to the method of Example 1), the VE group and the mupirocin group were applied with corresponding doses of drugs; the blank control group and the model group were applied with 0.5% NaCl solution once a day; the administration was continued for 21 days.

[0112] The common dominant bacteria on the skin include Acidobacteriota, unclassified_BacteriaChloroflexi, Myxococcota, Gemmatimonadota, Actinobacteriota, Nitrospirota, Verrucomicrobiota, Thermotogota, Planctomycetota, Unassigned, Desulfobacterota, Fibrobacterota, Spirochaetota, Proteobacteria, Patescibacteria, Bacteroidota, Firmicutes, Cyanobacteria, etc. Figure 4 It can be seen that the bacterial communities of Acidobacteriota, Unassigned, Desulfobacterota, Chloroflexi, Myxococcota, Gemmatimonadota, Actinobacteriota, Nitrospirota, Verrucomicrobiota and Thermotogota in the skin microecology of mice in the high, medium and low dose drug administration groups were significantly reduced compared with those in the model group. Compared with the model group, the concentrations of TNF-α, IL-4 and IL-13 in the serum of the normal group and each drug administration group were significantly lower than those in the model group. After the liposome gel prepared by the present invention improved the skin microecology of mice, the inflammatory factors were reduced, and the recovery of mouse specific dermatitis was faster.

[0113] The anti-inflammatory factors of the mice in the model group and the drug-treated group were detected by anti-inflammatory experiments. The concentrations of TNF-α, IL-4 and IL-13 in the serum of the mice treated with the gel were changed. Figure 4It can be seen that, compared with the blank group and the drug group, after 21 days of drug administration, the differences in the concentrations of TNF-α, IL-4, and IL-13 in the serum of the high-dose group and the medium-dose group were not significant; there were significant differences in the concentrations of TNF-α and IL-13 in the serum of the low-dose group (P<0.05, P<0.05), and there was no significant difference in the concentration of IL-4 (P=0.054); the concentration of TNF-α in the positive drug group was different from that of the blank group (P<0.05), and there was no difference in the concentration of IL-4 and IL-13 with the blank group. Compared with the model group, the concentrations of TNF-α, IL-4, and IL-13 in the serum of the normal group and each drug group were significantly lower than those of the model group (P<0.001), as shown in Table 17.

[0114] Table 17

[0115]

[0116] Note: The results are expressed as X±S, * compared with the model group, *: P<0.05, **: P<0.01, ***P<0.001; # compared with the positive drug group, #: P<0.05, ##: P<0.01, ###: P<0.001, n=6.

[0117] The skin antioxidant test was performed on the mice in the model group and the drug-treated group, as shown in Table 18.

[0118] Table 18

[0119]

[0120] Note: The results are expressed as X±S, * compared with the model group, *: P<0.05, **: P<0.01, ***P<0.001; # compared with the positive drug group, #: P<0.05, ##: P<0.01, ###: P<0.001, n=6.

[0121] As can be seen from Table 18, the SOD activities of mice in the high, medium and low drug administration groups, as well as the membrane lipid peroxidation index malondialdehyde MDA, indicate that the liposomes prepared by the present invention can maximize the antioxidant effect of bilberry on the skin.

[0122] The liposomes of the present invention were subjected to an in vitro antioxidant test (lyophilized powder), as Figure 5-7 As shown, the free radical scavenging rate of liposome gel Dpph, ABTS + The clearance rate and total reducing capacity are very strong, close to the antioxidant capacity of vitamin C.

[0123] Observation of skin tissue by Masson's trichrome staining:

[0124] Mouse skin tissue of about 1 cm×1 cm was taken and fixed in 4% paraformaldehyde for 24 hours, and the tissue was dehydrated, transparent, wax-impregnated, embedded, sliced ​​and baked. The slices were placed in xylene I and xylene II in turn, taken out after 10 minutes, and placed in different concentrations of ethanol and distilled water for 5 minutes each; stained with Weigert iron hematoxylin for 5-10 minutes, washed with water; differentiated with hydrochloric acid ethanol solution, washed with water; then blued with Masson blue solution, washed with water; then stained with Ponceau fuchsin staining solution for 5-10 minutes, washed with weak acid working solution prepared with distilled water and weak acid (2:1, v / v) for 1 minute; washed with phosphomolybdic acid solution for 1-2 minutes, and then washed with weak acid working solution for 1 minute; then directly placed in aniline blue staining solution for 1-2 minutes, and then washed with weak acid working solution for 1 minute; dehydrated quickly with 95% ethanol, and dehydrated quickly in anhydrous ethanol for 3 times; transparentized with xylene 3 times, each time for 1-2 minutes; finally sealed with neutral gum. The sealed slices were placed under a microscope to observe the morphological changes of skin collagen fibers, measure the area ratio of collagen fibers and analyze the data.

[0125] Each group of Masson-stained sections was photographed and observed under a 200x microscope. The results are as follows: Fig. 9 As shown in Table 19, the results of the measurement of the proportion of skin collagen fibers are shown in Table 19. It can be seen that the collagen fibers are blue, and the collagen fibers in the dermis of the blank group mice are of moderate thickness, compact and orderly, and the collagen fiber content is 39.15%; while the collagen fibers in the skin of the model group and the gel matrix group mice are disorderly, loose and missing, which is about 14% lower than that of the blank group, indicating that the formation process of atopic dermatitis destroys the collagen fibers in the skin. Compared with the model group, the collagen fiber content of the skin of the low-dose group mice increased by about 6%; the collagen fiber content of the skin of the medium-dose group and the high-dose group mice increased by about 9%; the collagen fiber content of the skin of the VE positive control group mice increased by about 10%. In general, the intervention of external application of drugs and VE can significantly reduce the damage to skin collagen fibers during the formation of atopic dermatitis, increase the proportion of collagen fibers in the skin, and have a certain anti-skin aging effect, and the high-dose group and the positive drug VE have the best effect. Since the effect of the liposome prepared by the present invention on increasing the content of collagen fibers is close to the anti-oxidation effect of the positive drug group, and the repairing power is close to the positive drug vitamin E, it is a liposome gel for natural skin repair and anti-oxidation.

[0126] Table 19

[0127]

[0128] The red bean and bilberry liposomes of the present invention can also be used to prepare a liposome cream, which comprises: 12g of stearic acid, 3.5g of glyceryl monostearate, 0.1g of ethylparaben, 1g of vaseline, 6g of liquid paraffin, 5g of glycerol, 0.4g of triethanolamine and 1g of red bean and bilberry liposomes, and water is added to make up to 100g.

[0129] Although the present invention is disclosed as above, the protection scope of the present invention is not limited thereto. Those skilled in the art may make various changes and modifications without departing from the spirit and scope of the present invention, and these changes and modifications will fall within the protection scope of the present invention.

Claims

1. An anti-oxidative and anti-aging red bean and bilberry liposome gel for rapidly repairing skin collagen fibers damaged by atopic dermatitis, characterized in that: The invention comprises red bean and cranberry liposome and gel, wherein every 100 ml of the red bean and cranberry liposome is dispersed in 15.5-133 g of gel.

2. The anti-oxidative and anti-aging red bean and bilberry liposome gel for rapidly repairing skin collagen fibers damaged by atopic dermatitis according to claim 1, characterized in that: The raw materials of the red bean and cranberry liposomes include red bean and cranberry polyphenols, soybean lecithin, cholesterol and a surfactant. The soybean lecithin and cholesterol form a lipid film that covers the surface of the red bean and cranberry polyphenols.

3. The anti-oxidative and anti-aging red bean and bilberry liposome gel for rapidly repairing skin collagen fibers damaged by atopic dermatitis according to claim 2, characterized in that: The mass ratio of soybean lecithin to cholesterol in the lipid film is 10:3, and Tween 80 is used as the surfactant.

4. The anti-oxidative and anti-aging red bean and bilberry liposome gel for rapidly repairing skin collagen fibers damaged by atopic dermatitis according to claim 3, characterized in that: The gel comprises 0.5-5 parts of poloxamer 188, 12-125 parts of poloxamer 407 and 3 parts of propylene glycol in parts by mass.

5. A method for preparing an anti-oxidative and anti-aging red bean and bilberry liposome gel for rapidly repairing skin collagen fibers damaged by atopic dermatitis, characterized in that: The steps include: Step 1, washing fresh blueberry, adding into ethanol solution for ultrasonic extraction, and obtaining blueberry ethanol extract; Step 2, enriching and purifying the bilberry polyphenols in the bilberry alcohol extract to obtain a bilberry polyphenol extract; Step 3, freeze-drying the blueberry polyphenol extract to obtain blueberry freeze-dried powder; Step 4: ① dissolving soybean lecithin and cholesterol in chloroform, and then evaporating the mixture to obtain a lipid film; ② adding freeze-dried red bean and blueberry powder and a surfactant to water and mixing the mixture with the lipid film, and then performing rotary evaporation and shaking at 30-40° C. to obtain a red bean and blueberry liposome suspension; ③ ultrasonically treating the red bean and blueberry liposome suspension, filtering the mixture, and granulating the mixture to obtain red bean and blueberry liposomes; Step 5: Evenly mix poloxamer 188, poloxamer 407 and propylene glycol according to their weight proportions to obtain a gel, add the gel to the red bean and bilberry liposomes, mix well, and let stand to obtain the red bean and bilberry liposome gel.

6. The method for preparing the anti-oxidative and anti-aging red bean and bilberry liposome gel for rapidly repairing skin collagen fibers damaged by atopic dermatitis according to claim 5, characterized in that: The concentration of the ethanol solution in step 1 is 75%, the heating time is 25min to 45min under the condition of ultrasonic power of 600W to 800W, the temperature is 55°C, and the material-liquid ratio is 1:3-1:

4.

7. The method for preparing the anti-oxidative and anti-aging red bean and bilberry liposome gel for rapidly repairing skin collagen fibers damaged by atopic dermatitis according to claim 6, characterized in that: In step 2, YWD07 macroporous resin is used to enrich and purify red bean bilberry polyphenols, specifically: wet column packing, distilled water compacting the adsorption column, static adsorption for 12h-18h, then washing the column with distilled water for chromatography, removing sugars and small molecules in the crude extract, and finally eluting with 80%-90% ethanol to obtain red bean bilberry polyphenol extract.

8. The method for preparing the anti-oxidative and anti-aging red bean and bilberry liposome gel for rapidly repairing skin collagen fibers damaged by atopic dermatitis according to claim 7, characterized in that: The freeze-drying time in step 3 is 24h-36h.

9. The method for preparing the anti-oxidative and anti-aging red bean and bilberry liposome gel for rapidly repairing skin collagen fibers damaged by atopic dermatitis according to claim 8, characterized in that: In step 5, 15.5 to 133 g of gel is added to every 100 ml of red bean and blueberry liposomes.

10. The method for preparing the anti-oxidative and anti-aging red bean and bilberry liposome gel for rapidly repairing skin collagen fibers damaged by atopic dermatitis according to claim 9, characterized in that: The standing time described in step 5 is not less than 24 hours.

Images