Preparation method of cell regeneration promoting emulsifier and application thereof, preparation method of cell regeneration promoting microemulsion

Abstract

The present invention relates to a preparation method of a cell regeneration promoting emulsifier and its application, and a preparation method of a cell regeneration promoting microemulsion. The preparation method of the cell regeneration promoting emulsifier of the present invention adopts aloe extract as a raw material, and the cell regeneration promoting emulsifier is prepared by the preparation method of the present invention, which has a good promoting effect on wound repair, can increase the expression of the pro-regeneration macrophage phenotype, and at the same time can enhance the stemness of hair follicle stem cells, which is conducive to the regeneration of hair follicles and their accessory organs, and finally realizes the regeneration of the damaged area tissue structure and function. The preparation method of the cell regeneration promoting emulsifier of the present invention has a simple production process, is safe and harmless, and a stable cell regeneration promoting emulsifier is prepared, which further improves the economic benefits, is suitable for large-scale production, and increases its market value. At the same time, the cell regeneration promoting emulsifier provides a new auxiliary means for repairing the skin and has good application prospects.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a preparation method of a cell regeneration promoting emulsifier and an application thereof, and a preparation method of a cell regeneration promoting microemulsion. Background Art

[0002] At present, there are many reasons for skin damage. According to surveys, about 26 million people in China suffer from burns of varying degrees every year, accounting for 2% of the total population, and the number of deaths is second only to traffic accidents. Among them, burns and scalds in children aged 0-12 account for 30%-50%, and this proportion is showing a continuous growth trend. In addition, trauma caused by genetic diseases and chronic trauma such as diabetes can also cause skin damage. Amputations caused by diabetic foot account for 27.3% of all amputations, and the 5-year mortality rate of patients after amputation is as high as 40.0%. Foot ulcers are common in diabetics. Due to the microenvironment of diabetics, this type of skin ulcer is difficult to heal and there is no better treatment. Therefore, many skin injuries in the human body cannot be restored by themselves, and large-area skin damage is very likely to cause infection and lead to death.

[0003] Therefore, cell regeneration promoting products have great application value in the field of skin repair. How to provide an effective and stable cell regeneration promoting product to assist in skin repair is of great significance. Summary of the invention

[0004] In order to overcome the shortcomings of the prior art, the first purpose of the present invention is to provide a method for preparing a cell regeneration emulsifier, which has a good promoting effect on wound repair, can increase the expression of pro-regeneration macrophage phenotype, and at the same time can enhance the stemness of hair follicle stem cells, which helps the regeneration of hair follicles and their appendages, and ultimately achieves the regeneration of tissue structure and function in the damaged area.

[0005] The second object of the present invention is to provide a method for preparing a microemulsion for promoting cell regeneration.

[0006] The third object of the present invention is to provide an application of an emulsifier for promoting cell regeneration.

[0007] One of the purposes of the present invention is achieved by adopting the following technical solution:

[0008] A method for preparing a cell regeneration promoting emulsifier comprises the following steps:

[0009] The aloe vera extract and wheat germ oil are mixed and dried, then reacted with pure water for homogenization, 95% ethanol by volume is added twice for mixed reaction and then dried, then mixed with pure water for homogenization for sterilization, and finally mixed with wheat germ oil for homogenization to obtain a cell regeneration promoting emulsifier.

[0010] Furthermore, the preparation method of the cell regeneration promoting emulsifier comprises the following steps:

[0011] 1) mixing aloe vera extract and wheat germ oil in a mass ratio of 1:(0.5-1.5) and drying the mixture to obtain a first mixture;

[0012] 2) mixing the first mixture of step 1) with pure water in a mass ratio of 1:(1.5-2.5), reacting the mixture, and then homogenizing the mixture to obtain a second mixture;

[0013] 3) mixing the second mixture in step 2) with 95% ethanol by volume in a volume ratio of 1:(1-4) to obtain a third mixture, and mixing the third mixture with 95% ethanol by volume in a volume ratio of 1:(1-4) to obtain a fourth mixture after drying;

[0014] 4) mixing the fourth mixture in step 3) with pure water in a mass ratio of 1:(10-14), homogenizing, continuing the reaction, and sterilizing to obtain a fifth mixture;

[0015] 5) The fifth mixture of step 4) is mixed with wheat germ oil in a volume ratio of 1:(0.1-0.45) and then homogenized to obtain a cell regeneration promoting emulsifier.

[0016] Furthermore, in step 1), the aloe vera extract and the wheat germ oil are mixed and then dried at 80-90° C. for 42-55 hours to obtain a first mixture.

[0017] Furthermore, in step 2), the first mixture is mixed with pure water and reacted at 80-90° C. for 20-28 hours, and then homogenized at 10,000 r / min for 8-12 minutes to obtain a second mixture.

[0018] Furthermore, in the step 3), the second mixture is mixed with 95% ethanol by volume and reacted at 55-65° C. for 1-4 hours; the third mixture is mixed with 95% ethanol by volume and reacted for 20-28 hours, and then dried at 55-65° C. for 42-55 hours to obtain a fourth mixture.

[0019] Furthermore, in the step 4), the fourth mixture is mixed with pure water and then homogenized at 10000 r / min for 3 to 8 minutes; the mixture is reacted at 85 to 95° C. for 2 to 24 hours; and the mixture is sterilized at 115 to 130° C. for 25 to 35 minutes to obtain a fifth mixture.

[0020] Furthermore, in the step 5), the fifth mixture is mixed with wheat germ oil and homogenized at 10000 r / min for 3 to 8 minutes to obtain a cell regeneration promoting emulsifier.

[0021] Furthermore, the preparation method of the aloe extract comprises the following steps:

[0022] The peeled aloe vera is ground into a gel, and the mixture is stirred at 12 to 20° C. for 20 to 28 hours to be liquefied and then filtered to obtain aloe vera liquid. The aloe vera liquid is then concentrated at 65 to 90° C. to obtain the aloe vera extract.

[0023] The second purpose of the present invention is achieved by adopting the following technical solution:

[0024] A method for preparing a microemulsion for promoting cell regeneration comprises the following steps:

[0025] Any of the above-mentioned cell regeneration promoting emulsifiers is mixed with silicone oil in a volume ratio of 1:(0.1-0.5) and then homogenized to obtain a cell regeneration promoting microemulsion.

[0026] Furthermore, the cell regeneration promoting emulsifier is mixed with silicone oil and homogenized at 10000 r / min for 3 to 8 minutes to obtain a cell regeneration promoting microemulsion.

[0027] The third object of the present invention is achieved by adopting the following technical solutions:

[0028] A use of a cell regeneration promoting emulsifier prepared by any of the above-mentioned preparation methods of a cell regeneration promoting emulsifier in repair and / or anti-inflammatory products.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] 1. The preparation method of the cell regeneration promoting emulsifier of the present invention adopts aloe extract as raw material, and the aloe extract is mixed with wheat germ oil and then dried, and then reacted with pure water for homogenization, and then added with 95% ethanol by volume twice for mixed reaction and dried, and then mixed with pure water for sterilization, and finally mixed with wheat germ oil for homogenization to obtain the cell regeneration promoting emulsifier, which has a good promoting effect on wound repair, can create a microenvironment conducive to the repair of damaged tissues, accelerate the repair of the pro-inflammatory period, can increase the expression of the pro-regeneration macrophage phenotype, and at the same time, it can enhance the stemness of hair follicle stem cells, which is conducive to the regeneration of hair follicles and their accessory organs, and finally realizes the regeneration of the tissue structure and function of the damaged area.

[0031] 2. The preparation method of the cell regeneration promoting emulsifier of the present invention has a simple production process, is safe and harmless, and produces a stable cell regeneration promoting emulsifier, which further improves the economic benefits, is suitable for large-scale production, and increases its market value. At the same time, the cell regeneration promoting emulsifier provides a new auxiliary means for repairing the skin and has good application prospects.

[0032] 3. The cell regeneration promoting microemulsion of the present invention has a good promoting effect on wound repair, can accelerate the repair of the pro-inflammatory period, increase the expression of the pro-regenerative macrophage phenotype, and achieve the regeneration of the damaged area tissue structure and function. The preparation method of the cell regeneration promoting microemulsion of the present invention has a simple preparation process and is suitable for large-scale production, which broadens the application scenarios of the cell regeneration promoting microemulsion and improves its market value. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The data graphs of tail fin length of zebrafish larvae at various time points in experimental group 1 and control group 1;

[0034] Figure 2 This is the data graph of the tail fin growth of zebrafish larvae in experimental group 1 and control group 1 every 24 hours;

[0035] Figure 3 This is the growth profile change diagram of zebrafish caudal fin in experimental group 1 and control group 1;

[0036] Figure 4 The data graphs show the regeneration area of ​​adult zebrafish in experimental group 2 and control group 2 at 1, 3, 5, 7, 9, 11, and 13 days after tail fin removal;

[0037] Figure 5 The growth profile of the tail fin of adult zebrafish in experimental group 2 and control group 2 on days 1, 7, and 11 after tail fin removal;

[0038] Figure 6 These are the wound pictures of rats in experimental group 3, experimental group 4 and control group 3 on days 0, 7, 14, 21 and 28 after scalding;

[0039] Figure 7 It is a quantitative analysis chart of wound healing rate of rats in experimental group 3, experimental group 4 and control group 3;

[0040] Figure 8 HE staining images of wound tissues of rats in experimental group 3, experimental group 4 and control group 3 on days 14 and 28;

[0041] Fig. 9 These are iNOS staining images of wound tissues of rats in experimental group 3, experimental group 4, and control group 3 on days 7 and 14;

[0042] Fig.10 These are Arg1 staining images of wound tissues of rats in experimental group 3, experimental group 4, and control group 3 on days 14 and 21;

[0043] Fig.11 These are the wound pictures of diabetic rats in experimental group 5, experimental group 6 and control group 4 on days 0, 3, 7 and 14;

[0044] Fig.12 It is a quantitative analysis chart of wound healing rate of diabetic rats in experimental group 5, experimental group 6 and control group 4;

[0045] Fig.13 HE staining images of diabetic rat tissues in experimental group 5, experimental group 6 and control group 4 on day 28;

[0046] Fig.14 This is the data graph of the number of hair follicles in the wound tissue of diabetic rats in experimental group 5, experimental group 6 and control group 4 on the 28th day. DETAILED DESCRIPTION

[0047] The technical solution of the present invention will be clearly and completely described below in conjunction with specific embodiments. Obviously, the described embodiments are only partial embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0048] A method for preparing a cell regeneration promoting emulsifier comprises the following steps:

[0049] The aloe vera extract and wheat germ oil are mixed and dried, then reacted with pure water for homogenization, 95% ethanol by volume is added twice for mixed reaction and then dried, then mixed with pure water for homogenization for sterilization, and finally mixed with wheat germ oil for homogenization to obtain a cell regeneration promoting emulsifier.

[0050] As one embodiment thereof, the method for preparing the cell regeneration promoting emulsifier comprises the following steps:

[0051] 1) mixing aloe vera extract and wheat germ oil in a mass ratio of 1:(0.5-1.5) and drying the mixture to obtain a first mixture;

[0052] 2) mixing the first mixture of step 1) with pure water in a mass ratio of 1:(1.5-2.5), reacting the mixture, and then homogenizing the mixture to obtain a second mixture;

[0053] 3) mixing the second mixture in step 2) with 95% ethanol by volume in a volume ratio of 1:(1-4) to obtain a third mixture, and mixing the third mixture with 95% ethanol by volume in a volume ratio of 1:(1-4) to obtain a fourth mixture after drying;

[0054] 4) mixing the fourth mixture in step 3) with pure water in a mass ratio of 1:(10-14), homogenizing, continuing the reaction, and sterilizing to obtain a fifth mixture;

[0055] 5) The fifth mixture of step 4) is mixed with wheat germ oil in a volume ratio of 1:(0.1-0.45) and then homogenized to obtain a cell regeneration promoting emulsifier.

[0056] The preparation method of the cell regeneration promoting emulsifier of the present invention adopts aloe extract as a raw material. The cell regeneration promoting emulsifier is prepared by the preparation method of the present invention, and the stability and bioaccessibility of the polysaccharide in the aloe extract are improved. The emulsifier has a good promoting effect on wound repair, can create a microenvironment conducive to the repair of damaged tissues, accelerate the repair of the pro-inflammatory period, and can increase the expression of the pro-regeneration macrophage phenotype. At the same time, the emulsifier can enhance the stemness of hair follicle stem cells, which is conducive to the regeneration of hair follicles and their appendages, and finally realizes the regeneration of the damaged area to prevent the structure and function.

[0057] As one embodiment, the mass ratio of the aloe vera extract to the wheat germ oil in step 1) is 1:1;

[0058] The mass ratio of the first mixture to pure water in step 2) is 1:2;

[0059] The volume ratio of the second mixture in step 3) to 95% ethanol is 1:2;

[0060] The volume ratio of the third mixture in step 3) to 95% ethanol is 1:2;

[0061] The mass ratio of the fourth mixture in step 4) to pure water is 1:12;

[0062] The volume ratio of the fifth mixture in step 5) to the wheat germ oil is 1:0.275.

[0063] As one embodiment, in step 1), the aloe extract and wheat germ oil are mixed and then dried at 80-90° C. for 42-55 hours to obtain the first mixture. Preferably, in step 1), the aloe extract and wheat germ oil are mixed and then dried at 85° C. for 48 hours to obtain the first mixture.

[0064] As one embodiment, in step 2), the first mixture is mixed with pure water, reacted at 80-90°C for 20-28h, and homogenized at 10000 r / min for 8-12min to obtain a second mixture. Preferably, in step 2), the first mixture is mixed with pure water, reacted at 85°C for 24h, and homogenized at 10000 r / min for 10min to obtain a second mixture.

[0065] As one embodiment, in step 3), the second mixture is mixed with 95% ethanol by volume and reacted at 55-65°C for 1-4 hours; the third mixture is mixed with 95% ethanol by volume and reacted for 20-28 hours, and then dried at 55-65°C for 42-55 hours to obtain a fourth mixture. Preferably, in step 3), the second mixture is mixed with 95% ethanol by volume and reacted at 60°C for 2 hours; the third mixture is mixed with 95% ethanol by volume and reacted for 24 hours, and then dried at 60°C for 48 hours to obtain a fourth mixture.

[0066] As one embodiment, in step 4), the fourth mixture is mixed with pure water and then homogenized at 10,000 r / min for 3 to 8 minutes; the mixture is reacted at 85 to 95°C for 2 to 24 hours; and the mixture is sterilized at 115 to 130°C for 25 to 35 minutes to obtain a fifth mixture. Preferably, in step 4), the fourth mixture is mixed with pure water and then homogenized at 10,000 r / min for 5 minutes; the mixture is reacted at 90°C for 12 hours; and the mixture is sterilized at 121°C for 30 minutes to obtain a fifth mixture.

[0067] As one embodiment, in step 5), the fifth mixture is mixed with wheat germ oil and homogenized at 10,000 r / min for 3 to 8 minutes to obtain the cell regeneration promoting emulsifier. Preferably, in step 5), the fifth mixture is mixed with wheat germ oil and homogenized at 10,000 r / min for 5 minutes to obtain the cell regeneration promoting emulsifier.

[0068] As one embodiment, the aloe extract is prepared from Aloe vera; the aloe extract contains purified polysaccharide ABPA2, and its structural formula is as follows:

[0069]

[0070] Wherein, R is one of H or acetyl group.

[0071] As one embodiment, the aloe extract is prepared from Aloe vera; the preparation method of the aloe extract comprises the following steps:

[0072] The peeled aloe vera is ground into a gelatinous state, and the mixture is continuously stirred at 12 to 20° C. for 20 to 28 hours for liquefaction, and then filtered to obtain aloe vera liquid, and then the aloe vera liquid is concentrated at 65 to 90° C. to obtain the aloe vera extract. Preferably, the peeled aloe vera is ground into a gelatinous state, and the mixture is continuously stirred at 15 to 17° C. for 24 hours for liquefaction, and then filtered to obtain aloe vera liquid, and then the aloe vera liquid is concentrated at 65 to 90° C. to obtain 5% of the volume of the original aloe vera liquid to obtain the aloe vera extract.

[0073] The present invention also provides a method for preparing a microemulsion for promoting cell regeneration, comprising the following steps:

[0074] Any of the above-mentioned cell regeneration promoting emulsifiers is mixed with silicone oil in a volume ratio of 1:(0.1-0.5) and then homogenized to obtain a cell regeneration promoting microemulsion.

[0075] As one embodiment, the volume ratio of the cell regeneration promoting emulsifier to the silicone oil is 1:0.3.

[0076] As one embodiment, the cell regeneration promoting emulsifier is mixed with silicone oil and homogenized at 10000r / min for 3 to 8 minutes to obtain a cell regeneration promoting microemulsion. Preferably, the cell regeneration promoting emulsifier is mixed with silicone oil and homogenized at 10000r / min for 5 minutes to obtain a cell regeneration promoting microemulsion. Wherein, the cell regeneration promoting microemulsion is ABPA-R5.

[0077] The present invention also provides use of a cell regeneration promoting emulsifier prepared by any of the above-mentioned methods for preparing the cell regeneration promoting emulsifier in repair and / or anti-inflammatory products.

[0078] The preparation method and application of the cell regeneration promoting emulsifier of the present invention are described below with specific examples.

[0079] Embodiment 1:

[0080] A method for preparing a cell regeneration promoting emulsifier comprises the following steps:

[0081] 1) mixing aloe vera extract and wheat germ oil in a mass ratio of 1:1 and drying the mixture at 85° C. for 48 hours to obtain a first mixture;

[0082] 2) mixing the first mixture of step 1) with pure water in a mass ratio of 1:2, reacting at 85° C. for 24 hours, and continuing to homogenize at 10000 r / min for 10 minutes to obtain a second mixture;

[0083] 3) mixing the second mixture in step 2) with 95% ethanol by volume in a volume ratio of 1:2 and reacting at 60° C. for 2 h to obtain a third mixture; mixing the third mixture with 95% ethanol by volume in a volume ratio of 1:2 and reacting for 24 h, and then continuing to dry at 60° C. for 48 h to obtain a fourth mixture;

[0084] 4) mixing the fourth mixture in step 3) with pure water at a mass ratio of 1:12, homogenizing at 10000 r / min for 5 min; continuing to react at 90° C. for 12 h; and sterilizing at 121° C. for 30 min to obtain a fifth mixture;

[0085] 5) The fifth mixture of step 4) and wheat germ oil were mixed at a volume ratio of 1:0.275 and homogenized at 10000 r / min for 5 min to obtain a cell regeneration promoting emulsifier.

[0086] Example 2

[0087] A method for preparing a cell regeneration promoting emulsifier comprises the following steps:

[0088] 1) mixing aloe vera extract and wheat germ oil in a mass ratio of 1:0.5 and drying the mixture at 80° C. for 55 h to obtain a first mixture;

[0089] 2) mixing the first mixture of step 1) with pure water in a mass ratio of 1:2.5, reacting at 80° C. for 28 hours, and continuing to homogenize at 10000 r / min for 8 minutes to obtain a second mixture;

[0090] 3) mixing the second mixture in step 2) with 95% ethanol by volume in a volume ratio of 1:1, reacting at 55° C. for 4 hours to obtain a third mixture; mixing the third mixture with 95% ethanol by volume in a volume ratio of 1:4, reacting for 28 hours, and then continuing to dry at 65° C. for 42 hours to obtain a fourth mixture;

[0091] 4) mixing the fourth mixture in step 3) with pure water at a mass ratio of 1:10, homogenizing at 10000 r / min for 3 min; continuing to react at 85° C. for 24 h; sterilizing at 130° C. for 25 min to obtain a fifth mixture;

[0092] 5) The fifth mixture of step 4) was mixed with wheat germ oil at a volume ratio of 1:0.45 and homogenized at 10000 r / min for 8 min to obtain a cell regeneration promoting emulsifier.

[0093] Example 3

[0094] A method for preparing a cell regeneration promoting emulsifier comprises the following steps:

[0095] 1) mixing aloe vera extract and wheat germ oil in a mass ratio of 1:1.5 and drying the mixture at 90° C. for 42 hours to obtain a first mixture;

[0096] 2) mixing the first mixture of step 1) with pure water at a mass ratio of 1:1.5, reacting at 90° C. for 20 h, and continuing to homogenize at 10000 r / min for 12 min to obtain a second mixture;

[0097] 3) mixing the second mixture in step 2) with 95% ethanol by volume in a volume ratio of 1:4, reacting at 65° C. for 1 h to obtain a third mixture, mixing the third mixture with 95% ethanol by volume in a volume ratio of 1:1 for 20 h, and then continuing to dry at 55° C. for 55 h to obtain a fourth mixture;

[0098] 4) mixing the fourth mixture in step 3) with pure water at a mass ratio of 1:14, homogenizing at 10000 r / min for 8 min; continuing to react at 95° C. for 2 h; and sterilizing at 115° C. for 35 min to obtain a fifth mixture;

[0099] 5) The fifth mixture of step 4) was mixed with wheat germ oil at a volume ratio of 1:0.1 and homogenized at 10000 r / min for 3 min to obtain a cell regeneration promoting emulsifier.

[0100] Example 4

[0101] A method for preparing a microemulsion for promoting cell regeneration comprises the following steps:

[0102] The cell regeneration promoting emulsifier prepared in Example 1 was mixed with silicone oil in a volume ratio of 1:0.3 and homogenized at 10000 r / min for 5 min to obtain a cell regeneration promoting microemulsion.

[0103] Comparative Example 1

[0104] 1) mixing aloe vera extract and wheat germ oil in a mass ratio of 1:1 and drying the mixture at 85° C. for 48 hours to obtain a first mixture;

[0105] 2) mixing the first mixture of step 1) with pure water in a mass ratio of 1:2, reacting at 85° C. for 24 hours, and continuing to homogenize at 10000 r / min for 10 minutes to obtain a second mixture;

[0106] 3) mixing the second mixture in step 2) with 95% ethanol by volume in a volume ratio of 1:4 and reacting at 60° C. for 2 h to obtain a third mixture, and extracting ethanol from the third mixture by a rotary evaporator to obtain a fourth mixture;

[0107] 4) The fourth mixture of step 3) is mixed with pure water in a mass ratio of 1:12, and the mixture is homogenized at 10000 r / min for 5 minutes to obtain a fifth mixture; 5) The fifth mixture of step 4) ABPA2 is mixed with wheat germ oil in a volume ratio of 1:0.275, and the mixture is homogenized at 10000 r / min for 5 minutes to obtain a cell regeneration promoting emulsifier, and the cell regeneration promoting emulsifier is mixed with silicone oil in a volume ratio of 1:0.3, and the mixture is homogenized at 10000 r / min for 5 minutes to obtain an emulsion.

[0108] Comparative Example 2

[0109] 1) mixing aloe vera extract and wheat germ oil in a mass ratio of 1:1 and drying the mixture at 85° C. for 48 hours to obtain a first mixture;

[0110] 2) mixing the first mixture of step 1) with pure water in a mass ratio of 1:2, reacting at 85° C. for 24 hours, and continuing to homogenize at 10000 r / min for 10 minutes to obtain a second mixture;

[0111] 3) mixing the second mixture in step 2) with 95% ethanol by volume in a volume ratio of 1:2 and reacting at 60° C. for 2 h to obtain a third mixture; mixing the third mixture with 95% ethanol by volume in a volume ratio of 1:2 and reacting for 24 h; and then extracting ethanol with a rotary evaporator to obtain a fourth mixture;

[0112] 4) mixing the fourth mixture in step 3) with pure water at a mass ratio of 1:12, homogenizing at 10000 r / min for 5 min; continuing to react at 90° C. for 12 h; and sterilizing at 121° C. for 30 min to obtain a fifth mixture;

[0113] 5) The fifth mixture of step 4) is mixed with wheat germ oil at a volume ratio of 1:0.275, and then homogenized at 10000 r / min for 5 min to obtain a cell regeneration promoting emulsifier. The cell regeneration promoting emulsifier is mixed with silicone oil at a volume ratio of 1:0.3, and then homogenized at 10000 r / min for 5 min to obtain an emulsion.

[0114] Performance Testing

[0115] 1. Stability and fluidity test

[0116] After the examples 1-4 and the comparative examples 1-2 were placed for 60 hours, the samples were observed, and the stability and fluidity of the samples were judged by sensory organs. The results are shown in Table 1.

[0117] Table 1 Stability and fluidity test results of Examples 1-4 and Comparative Examples 1-2

[0118]

[0119] 2. Zebrafish larvae experiment - efficacy test of microemulsion promoting cell regeneration

[0120] 10 zebrafish fry with a weight difference of no more than 10g were selected and randomly divided into two groups. The two groups of zebrafish fry were cultured in two culture water tanks with a pH of 7.2 and a water temperature of 28°C, and the tail fins of the two groups of zebrafish fry were removed. The first group of zebrafish fry with the tail fin removed was not treated and continued to be cultured in the culture water tank as control group 1. The second group of zebrafish fry with the tail fin removed was cultured in a culture water tank containing the cell regeneration promoting microemulsion of Example 4 with a concentration of 0.0375%, as experimental group 1. The growth changes of the wound of the zebrafish fry after the tail fin was cut were observed under a microscope, and the observation and photography were recorded every 4 hours, and the length of the tail fin growing every day after cutting was counted.

[0121] After the tail fin was removed, the growth of the tail fins of the experimental group 1 and the control group 1 was observed:

[0122] (1) The tail fin lengths of the two groups of zebrafish larvae at different time points were recorded. Figure 1 As shown;

[0123] (2) The length of the tail fin growth of the two groups of zebrafish larvae was recorded every 24 hours. The results are as follows: Figure 2 As shown;

[0124] (3) Record the changes in the tail fin growth profiles of the two groups of zebrafish larvae. Figure 3 shown.

[0125] 3. Adult zebrafish experiment - efficacy test of microemulsion promoting cell regeneration

[0126] Ten 3-month-old adult zebrafish with a weight difference of no more than 10 g were selected and randomly divided into two groups. The two groups of adult zebrafish were cultured in two culture water tanks with a pH of 7.2 and a water temperature of 28°C, and the tail fins of the two groups of adult zebrafish were cut off by 1 / 2. The first group of adult zebrafish with the tail fin cut off were not treated and continued to be cultured in the culture water tank as the control group 2. The second group of zebrafish fry with the tail fin cut off were cultured in a culture water tank containing the cell regeneration promoting microemulsion of Example 4 at a concentration of 0.01%, as the experimental group 2. The growth changes of the wound of the adult zebrafish after the tail fin was cut were observed under a microscope, and the area of ​​regeneration per day after the tail fin was cut off was counted.

[0127] After the tail fin was removed, the growth of the tail fins of the experimental group 2 and the control group 2 was observed:

[0128] (1) Record the regeneration area 1, 3, 5, 7, 9, 11, and 13 days after tail fin removal. Figure 4 As shown;

[0129] (2) The growth profile of the caudal fin of two groups of adult zebrafish was recorded on days 1, 7, and 11 after caudal fin removal. Figure 5 shown.

[0130] 4. Rat experiment - efficacy test of cell regeneration microemulsion

[0131] 15 healthy rats with a weight difference of no more than 20g were selected and randomly divided into three groups. A constant pressure and constant temperature scald instrument was used to make a rat deep second degree scald model, so that the scald site was a deep second degree scald. After 24 hours of observation, the wound surface showed obvious inflammatory changes, such as purple red congestion, edema, etc. The area and depth of scalds in rats in each group were similar, and the wound area of ​​the rats was recorded. After the animal model was prepared, each group was caged, and the wound of the first group of rats was not treated as control group 3. The second group of rats was applied with 0.5mL of smear every day on the wound, using the cell regeneration promoting microemulsion of Example 4, as experimental group 3. After 7 days, the third group of rats was applied with 0.5mL of smear every day on the wound using the cell regeneration promoting microemulsion of Example 4, as experimental group 4. The growth changes of the wounds of the three groups of rats were observed.

[0132] (1) Record the wound images of each group on days 0, 7, 14, 21, and 28 after burns. Figure 6 As shown;

[0133] (2) Quantitative analysis of wound healing rate of each group was recorded. The results are as follows: Figure 7 As shown;

[0134] (3) M1 (pro-inflammatory) and M2 (pro-regenerative) macrophage markers (iNOS and Arg1 for immunohistochemistry and CD86 and CD206 for immunofluorescence) were selected to evaluate the effect of the pro-cell regeneration microemulsion on the development of inflammation during wound healing. HE staining images of the wound tissues of each group were recorded on days 14 and 28. The results are shown in Table 1. Figure 8 As shown;

[0135] (4) Record the iNOS staining images of wound tissues in each group on days 7 and 14. Fig. 9 As shown;

[0136] (5) Record the Arg1 staining images of wound tissues in each group on days 14 and 21. Fig.10 shown.

[0137] 5. Diabetic rat experiment - efficacy test of cell regeneration microemulsion

[0138] 15 diabetic rats with a weight difference of no more than 20g were selected and randomly divided into three groups. A constant pressure and constant temperature scald instrument was used to make a rat deep second degree scald model, so that the scald site was a deep second degree scald. After 24 hours of observation, the wound surface showed obvious inflammatory changes, such as purple red congestion, edema, etc. The area and depth of scalds in rats in each group were similar, and the wound area of ​​the rats was recorded. After the animal model was prepared, each group was caged, and the wound of the first group of rats was not treated, as control group 4. The second group of rats was applied with 0.5mL of the cell regeneration promoting microemulsion of Example 4 every day at the wound, as experimental group 5. After 6 days, the third group of rats was applied with 0.5mL of the cell regeneration promoting microemulsion of Example 4 every day at the wound, as experimental group 6. The growth changes of the wounds of the three groups of rats were observed.

[0139] (1) Record the wound surface images of each group on days 0, 3, 7, and 14 after burns, such as Fig.11 As shown;

[0140] (2) Quantitative analysis of wound healing rate of each group was recorded. The results are as follows: Fig.12 As shown;

[0141] (3) M1 (pro-inflammatory) and M2 (pro-regenerative) macrophage markers (iNOS and Arg1 for immunohistochemistry and CD86 and CD206 for immunofluorescence) were selected to evaluate the effect of the pro-cell regeneration microemulsion on the development of inflammation during wound healing. The HE staining images of the wound tissues of each group were recorded on the 28th day. The results are shown in Table 1. Fig.13 As shown;

[0142] (4) Count the number of hair follicles in the wound tissue of each group on the 28th day. The results are as follows: Fig.14 shown.

[0143] Depend on Figure 1 It can be seen that 48 hours after the tail fin was removed, the average growth length of the tail fin of the zebrafish fry in the experimental group 1 with the cell regeneration promoting microemulsion of Example 4 having the tail fin removed was significantly greater than that of the control group 1, and at the 72nd and 120th hours after the tail fin was removed, the average growth length of the zebrafish fry in the experimental group 1 was still significantly greater than that of the control group 1.

[0144] Depend on Figure 2 It can be seen that the length of the tail fin growth of the two groups of zebrafish larvae was recorded every 24 hours. The regeneration rate of the control group 1 reached its peak at the 72nd hour after the tail fin was cut off, while the regeneration rate of the tail fin of the experimental group 1 reached its peak at the 48th hour after treatment, and the peak regeneration rate of the experimental group 1 was higher than that of the control group 1. Figure 3It can be seen that the tail fin of the zebrafish larvae in the experimental group 1 has basically completed regeneration within 48 hours after tail amputation; while the tail fin of the zebrafish larvae in the control group 1 has basically completed regeneration within 72 hours after tail amputation.

[0145] Depend on Figure 4 It can be seen that the caudal fin regeneration area of ​​adult zebrafish in experimental group 2 is generally higher than that in control group 2, and the difference is most significant on the seventh day. Figure 7 It can be seen that on the 14th day, the cure rate of experimental group 3 was 52.28±2.83%, the cure rate of experimental group 4 was 49.59±5.58%, and the cure rate of control group 3 was 36.24±7.30%. Obviously, the cure rates of experimental group 3 and experimental group 4 were significantly higher than those of control group 3. On the 21st day, the wound healing rates of experimental group 3 and experimental group 4 were 70.48±2.48% and 67.38±7.2%, respectively, which were significantly higher than the healing rate of control group 3, which was 59.64±2.58%. Figure 8 It can be seen that after 14 days of treatment, a large number of inflammatory cells infiltrated the wound surface and adjacent areas in each group, and collagen was deposited. However, unlike the control group 3, the collagen near the wound surface in the experimental groups 3 and 4 was being decomposed by inflammatory cells. With the further development of the healing process, the inflammatory response in the control group 3 was alleviated on the 28th day, but the collagen still existed. In the experimental groups 3 and 4, the epidermis and dermis were fully formed, and there was no obvious inflammatory cell infiltration.

[0146] Depend on Fig. 9 It can be seen that compared with the 7th day, the iNOS levels in experimental group 3, experimental group 4 and control group 3 on the 14th day showed a downward trend, indicating that the acute inflammation caused by the injury gradually subsided. Fig.10 It can be seen that the control group 3 had only a small number of Arg 1 positive cells on the 14th day, and even fewer on the 21st day. However, the experimental groups 3 and 4 had more Arg1 positive cells during the same period, indicating that the wounds of the rats treated with the cell regeneration-promoting microemulsion smoothly entered the pro-regeneration stage from the pro-inflammatory stage. Fig.11 , Fig.12 and Fig.13 It can be seen that the wound healing of rats in experimental groups 5 and 6 was significantly accelerated after 7 days, while the wound healing speed of rats in control group 4 was significantly slow. Fig.14 It can be seen that the number of hair follicles in the wound area of ​​diabetic rats in each group was counted. The number of hair follicles in the wound area of ​​diabetic rats in experimental groups 5 and 6 was significantly higher than that in control group 4. The wound surface smoothly entered the pro-regeneration period from the pro-inflammatory period. When the regeneration ability is impaired, the cell regeneration microemulsion also has a regeneration-promoting effect.

[0147] Therefore, the cell regeneration promoting emulsifier of the present invention adopts aloe extract as raw material, and the cell regeneration promoting emulsifier is prepared by the preparation method of the present invention, which can be applied in repair and / or anti-inflammatory products, and has a good promoting effect on wound repair, can create a microenvironment conducive to the repair of damaged tissue, accelerate the repair of the pro-inflammatory period, and can increase the expression of the pro-regeneration macrophage phenotype. At the same time, it can enhance the dryness of hair follicle stem cells, help the regeneration of hair follicles and their accessory organs, and finally achieve the regeneration of damaged regional tissue structure and function. The preparation method of the cell regeneration promoting emulsifier of the present invention has a simple production process, is safe and harmless, and a stable cell regeneration promoting emulsifier is prepared, which further improves the economic benefit, is suitable for large-scale production, and increases its market value. At the same time, the cell regeneration promoting emulsifier provides a new auxiliary means for repairing the skin, has a good promoting effect on wound repair, can create a microenvironment conducive to the repair of damaged tissue, and has the ability to promote cell regeneration. It has good application prospects.

[0148] For those skilled in the art, various other corresponding changes and deformations can be made according to the technical solutions and concepts described above, and all of these changes and deformations should fall within the protection scope of the claims of the present invention.

Claims

1. A method for preparing a cell regeneration-promoting emulsion, characterized in that: The following steps are involved: The aloe vera extract and wheat germ oil are mixed and dried, then reacted with pure water for homogenization, 95% ethanol by volume is added twice for mixed reaction, then dried, then mixed with pure water for homogenization for sterilization, and finally mixed with wheat germ oil for homogenization to obtain a cell regeneration promoting emulsion; The specific steps are as follows: 1) mixing aloe vera extract and wheat germ oil in a mass ratio of 1:(0.5-1.5) and drying at 80-90° C. for 42-55 hours to obtain a first mixture; 2) mixing the first mixture of step 1) with pure water in a mass ratio of 1:(1.5-2.5), reacting at 80-90° C. for 20-28 hours, and continuing to homogenize at 10000 r / min for 8-12 minutes to obtain a second mixture; 3) mixing the second mixture of step 2) with 95% ethanol by volume in a volume ratio of 1:(1-4) to obtain a third mixture; mixing the third mixture with 95% ethanol by volume in a volume ratio of 1:(1-4) to obtain a fourth mixture after drying at 55-65° C.; 4) mixing the fourth mixture in step 3) with pure water in a mass ratio of 1:(10-14), homogenizing, continuing the reaction, and sterilizing to obtain a fifth mixture; 5) mixing the fifth mixture of step 4) with wheat germ oil in a volume ratio of 1:(0.1-0.45) and homogenizing to obtain a cell regeneration promoting emulsion; Wherein, the preparation method of the aloe extract comprises the following steps: The peeled aloe vera is ground into a gel, and the mixture is stirred at 12 to 20° C. for 20 to 28 hours to be liquefied and filtered to obtain aloe vera liquid. The aloe vera liquid is then concentrated at 65 to 90° C. to obtain the aloe vera extract.

2. The method for preparing a cell regeneration promoting emulsion according to claim 1, characterized in that: In the step 3), the second mixture is mixed with 95% ethanol by volume and reacted at 55-65° C. for 1-4 hours; the third mixture is mixed with 95% ethanol by volume and reacted for 20-28 hours, and then dried at 55-65° C. for 42-55 hours to obtain a fourth mixture.

3. The method for preparing a cell regeneration promoting emulsion according to claim 1, characterized in that: In the step 4), the fourth mixture is mixed with pure water and homogenized at 10000 r / min for 3 to 8 minutes; the mixture is reacted at 85 to 95° C. for 2 to 24 hours; and the mixture is sterilized at 115 to 130° C. for 25 to 35 minutes to obtain a fifth mixture; In the step 5), the fifth mixture is mixed with wheat germ oil and homogenized at 10000 r / min for 3 to 8 minutes to obtain a cell regeneration promoting emulsion.

4. A method for preparing a microemulsion for promoting cell regeneration, characterized in that: The following steps are involved: The cell regeneration promoting emulsion prepared by the preparation method of the cell regeneration promoting emulsion according to any one of claims 1 to 3 is mixed with silicone oil at a volume ratio of 1:(0.1-0.5) and then homogenized to obtain a cell regeneration promoting microemulsion.

5. The method for preparing a cell regeneration promoting microemulsion according to claim 4, characterized in that: The cell regeneration promoting emulsion is mixed with silicone oil and homogenized at 10000r / min for 3 to 8 minutes to obtain the cell regeneration promoting microemulsion.

6. Use of the cell regeneration promoting emulsion prepared by the preparation method of the cell regeneration promoting emulsion according to any one of claims 1 to 3 in the preparation of skin repair and / or anti-inflammatory products.

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