Stearic acid modified cistanche polysaccharide entrapped bakuchiol micelle and application thereof

Abstract

The invention relates to the technical field of medicine, in particular to stearic acid modified cistanche polysaccharide entrapped bakuchiol micelles and application thereof.The preparation method of the micelles comprises the steps that under nitrogen protection, stearic acid, 4-dimethylaminopyridine and 1-ethyl-(3-dimethylaminopropyl) carbodiimide are dissolved in anhydrous dimethyl sulfoxide, stirring is conducted, and a mixture is obtained; stirring and activating carboxyl at room temperature to obtain a mixed solution; dissolving cistanche deserticola polysaccharide in anhydrous dimethyl sulfoxide to obtain a dissolved cistanche deserticola polysaccharide solution, dropwise adding the mixed solution into the cistanche deserticola polysaccharide solution, and stirring at room temperature for a period of time for reaction to obtain a reaction product; carrying out alcohol precipitation and dialysis on the reaction product to obtain a product; drying the product to obtain the stearic acid modified cistanche deserticola polysaccharide; the stearic acid modified cistanche polysaccharide and bakuchiol are mixed and subjected to ultrasonic treatment to obtain the stearic acid modified cistanche polysaccharide entrapped bakuchiol micelle. The micelle has good anti-aging, whitening and wound repairing effects.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and relates to a nano micelle for skin whitening and anti-aging effects and an application thereof, and specifically relates to a stearic acid-modified Cistanche deserticola polysaccharide-encapsulated bakuchiol micelle and an application thereof. Background Art

[0002] The biosynthesis of melanin in the skin is a reaction of hydroxylating tyrosine in the body under the action of tyrosinase or other substances. Among them, tyrosinase is a biologically functional copper-containing oxidase, and is also a key rate-limiting enzyme in melanin synthesis. It can hydroxylate L-tyrosine to L-dopa (with monophenolase activity), and then oxidize L-dopa to dopaquinone (with diphenolase activity). By inhibiting the activity of tyrosinase, targeting tyrosinase is the main direction of whitening cosmetics research and development. The main cause of skin aging is the combined effect of internal factors such as free radicals, collagen damage, etc., and external factors such as excessive exposure to sunlight, which cause the occurrence of phenomena including affecting cell activity, reducing collagen and elastin synthesis and increasing degradation, and ultimately leading to symptoms of thinning, drying, sagging, decreased elasticity, uneven pigmentation and deepening of wrinkles.

[0003] The epidermis is located in the outermost layer of the skin. From the outside to the inside, it includes five layers: the stratum corneum, the clear layer, the granular layer, the spinous layer and the basal layer. Among them, the stratum corneum is the main barrier for drugs to pass through the skin. It is composed of multiple layers of keratinocytes and is tightly embedded in each other. The gaps between the stratum corneum cells are filled with lipid substances, which can play a defensive role to protect the body from various physical, chemical, microbial and other external factors. Transdermal drug molecules mainly enter the deep layer of the skin by passing through the lipids between keratinocytes. The oil-water partition coefficient P of drug molecules is the main factor affecting the transdermal absorption and penetration process. It is generally believed that when logP<-2, the compound cannot pass through the lipid membrane because of its strong water solubility; when logP>3, the compound is difficult to be released from the membrane on the other side of the cell due to its strong fat solubility and enter the nearby blood vessels or lymphatic vessels; only drugs with a certain degree of water solubility and fat solubility can pass through the lipid area between cells more easily and achieve better skin absorption.

[0004] Bakuchiol is a monoterpene phenolic compound, mainly found in the leguminous plant Psoralea corylifolia. Bakuchiol can reduce the proliferation and production of melanin by inhibiting the synthesis of tyrosinase, thereby achieving a whitening effect. In addition, bakuchiol can also increase the levels of type I and type VII collagen to inhibit collagen degradation, thereby playing an anti-aging role. Because of its whitening and anti-aging effects, bakuchiol has been widely used in the field of cosmetics. However, bakuchiol is a fat-soluble drug with an oil-water partition coefficient logP of 4 to 5, which makes it difficult for it to pass through the lipid area between cells in the skin and cannot effectively reach the deep layer of the skin, thereby limiting its use as an external medicine in the treatment of skin diseases. Cistanche deserticola is a precious traditional Chinese medicinal material for both medicine and food in my country. Its application was first recorded in "Shennong's Herbal Classic" and is known as the "Desert Ginseng". The Cistanche deserticola polysaccharide contained therein is one of the main components of its whitening and anti-aging effects. Cistanche polysaccharide can increase the hydroxyproline content in animal skin cells, increase the collagen fiber content, improve skin elasticity, and thus delay skin aging. However, Cistanche polysaccharide is a water-soluble drug and is difficult to penetrate well into the skin layer, which seriously affects the skin absorption capacity of Cistanche polysaccharide.

[0005] Some common brand products in the cosmetics market, such as Kate Somerville Whitening and Moisturizing Repairing Facial Essence, Avene Moisturizing Lifting and Firming Eye Cream, REN Sensitive Skin Moisturizing and Soothing Repairing Essence, BIODERMA Oil Control Lotion, etc., all contain bakuchiol as an anti-aging ingredient. However, due to the fat solubility of bakuchiol, its role in cosmetics is greatly limited, and there is no report on the skin drug delivery system or cosmetics that use bakuchiol and Cistanche polysaccharide to work together. Summary of the invention

[0006] In view of the above problems, the object of the present invention is to provide a nano micelle system that can integrate fat-soluble bakuchiol and water-soluble Cistanche polysaccharide to jointly exert skin whitening and anti-aging effects. The micelle utilizes stearic acid to modify water-soluble Cistanche polysaccharide to make it amphiphilic, self-assemble to form micelles, and encapsulate fat-soluble drug bakuchiol inside the micelles. The bakuchiol micelles encapsulated by stearic acid-modified Cistanche polysaccharide prepared by the present invention can improve the solubility and bioavailability of bakuchiol, enhance its skin retention capacity, and improve the skin permeability of Cistanche polysaccharide at the same time, and further synergistically exert skin whitening and anti-aging effects.

[0007] Specifically, the technical solution of the present invention is as follows:

[0008] The first aspect of the present invention provides a method for preparing stearic acid-modified Cistanche deserticola polysaccharide-encapsulated bakuchiol micelles, comprising the following steps:

[0009] (1) Under nitrogen protection, stearic acid (SA), 4-dimethylaminopyridine (DMAP) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC·Cl) were dissolved in anhydrous dimethyl sulfoxide (DMSO), and the carboxyl groups were activated by stirring at room temperature to obtain a mixed solution;

[0010] (2) dissolving the Cistanche polysaccharide in anhydrous dimethyl sulfoxide (DMSO) to obtain a dissolved Cistanche polysaccharide solution, dropwise adding the mixed solution obtained in step (1) to the Cistanche polysaccharide solution, stirring at room temperature for a period of time to react, and obtaining a reaction product;

[0011] (3) after the reaction is completed, anhydrous ethanol is added to the reaction product for alcohol precipitation, after the alcohol precipitation is completed, the precipitate is collected, and the precipitate is dialyzed to obtain a dialyzed product;

[0012] (4) freeze-drying the dialyzed product to obtain stearic acid-modified Cistanche deserticola polysaccharide;

[0013] (5) dissolving the stearic acid-modified Cistanche polysaccharide in water to obtain solution 1, dissolving bakuchiol in ethyl acetate to obtain solution 2, mixing solution 1 and solution 2, and removing the ethyl acetate after ultrasonic treatment to obtain stearic acid-modified Cistanche polysaccharide-encapsulated bakuchiol micelles.

[0014] The stearic acid-modified Cistanche polysaccharide-encapsulated bakuchiol micelle drug delivery system obtained in the present application has the advantages of high osmotic concentration and small side effects. The micelles are supramolecular core-shell nanoparticles formed by self-assembly of an amphiphilic block copolymer after it is dissolved in water based on the action of non-covalent forces such as hydrophobic interactions, hydrogen bonds, and electrostatic interactions. The micelles are composed of a hydrophilic shell encapsulating a hydrophobic core. The stearic acid-modified Cistanche polysaccharide-encapsulated bakuchiol micelles have the advantages of high drug loading and good stability, and can improve the solubility of hydrophobic drugs, thereby improving the skin absorption capacity of hydrophobic drugs and enhancing the retention capacity of drugs in skin tissue.

[0015] In a specific embodiment, in step (1), the molar ratio of stearic acid, 4-dimethylaminopyridine and 1-ethyl-(3-dimethylaminopropyl)carbodiimide is: 0.01-1.0: 0.01-1.0: 0.01-1.0.

[0016] In a specific embodiment, the stirring time at room temperature in step (1) is 0.5 to 1 h.

[0017] In a specific embodiment, the stirring time at room temperature in step (2) is 46 to 50 hours;

[0018] Preferably, the stirring time at room temperature is 48 h.

[0019] In a specific embodiment, the step of alcohol precipitation in step (3) is: adding 8 to 12 times the amount of anhydrous ethanol to the reaction product for alcohol precipitation overnight, collecting the dialyzed product the next day and transferring it to a dialysis bag, changing the water in the morning and evening and dialyzing with distilled water for 46 to 50 hours;

[0020] Preferably, the dialysis time is 48h;

[0021] Preferably, the molecular weight cutoff of the dialysis bag is 10,000 to 14,000, and more preferably, the molecular weight cutoff of the dialysis bag is 12,000.

[0022] In a specific embodiment, in step (5), the mass ratio of the stearic acid-modified Cistanche deserticola polysaccharide to the bakuchiol is 9 to 11:1;

[0023] In a specific embodiment, the specific steps of ultrasonic treatment in step (5) are: ultrasonication for 10 minutes using an ultrasonic cell disruptor probe, preferably, the ultrasonic power is 500 W, the single ultrasonication time is 10 seconds, and the stop interval is 5 seconds.

[0024] In the second aspect of the present invention, provided is a stearic acid-modified Cistanche polysaccharide-encapsulated Bakuchiol micelle prepared by the method for preparing the stearic acid-modified Cistanche polysaccharide-encapsulated Bakuchiol micelle according to the first aspect.

[0025] In the third aspect of the present invention, there is provided a method for preparing the stearic acid-modified Cistanche polysaccharide-encapsulated bakuchiol micelles described in the first aspect and\or the use of the stearic acid-modified Cistanche polysaccharide-encapsulated bakuchiol micelles described in the second aspect in the preparation of medicines or cosmetics with whitening effects.

[0026] In the fourth aspect of the present invention, there is provided a method for preparing the stearic acid-modified Cistanche polysaccharide-encapsulated bakuchiol micelles described in the first aspect and\or the use of the stearic acid-modified Cistanche polysaccharide-encapsulated bakuchiol micelles described in the second aspect in the preparation of medicines or cosmetics with anti-aging effects.

[0027] In the fourth aspect of the present invention, there is provided a method for preparing the bakuchiol micelles encapsulated by the stearic acid-modified Cistanche polysaccharide described in the first aspect and\or the use of the bakuchiol micelles encapsulated by the stearic acid-modified Cistanche polysaccharide described in the second aspect in the preparation of medicines or cosmetics with wound repair efficacy.

[0028] The present invention has the following beneficial effects:

[0029] The stearic acid-modified Cistanche polysaccharide-encapsulated bakuchiol micelles prepared in the present invention have good whitening effects, anti-aging effects and wound repair effects, and can be used to prepare medicines or cosmetics with whitening effects, anti-aging effects and wound repair effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0031] Figure 1 The infrared characterization images of stearic acid, Cistanche deserticola polysaccharide and stearic acid-modified Cistanche deserticola polysaccharide in the present invention;

[0032] Figure 2 Figure 1 is a characterization diagram of the physicochemical properties of the stearic acid modified Cistanche polysaccharide-encapsulated Bakuchiol micelles in the present invention, Figure a is a Tyndall effect diagram, Figure b is a particle size diagram of the stearic acid modified Cistanche polysaccharide-encapsulated Bakuchiol micelles, and Figure c is the Zeta potential of the stearic acid modified Cistanche polysaccharide-encapsulated Bakuchiol micelles;

[0033] Figure 3 This is a diagram showing the distribution of fluorescence in cells in the present invention;

[0034] Figure 4 This is an analysis diagram of the quantitative analysis of the fluorescence intensity of the probe bound to the cells using a flow cytometer in the present invention;

[0035] Figure 5 This is a fluorescence microscope picture of the retention of micelles in the skin of the present invention;

[0036] Figure 6 The figure is the effect of three groups of samples of free bakuchiol, bakuchiol micelles and blank micelles on the survival rate of mouse melanoma cells (B16 cells) in the present invention;

[0037] Figure 7 This is a graph showing the effects of three groups of samples, namely, free bakuchiol, micelles of bakuchiol and blank micelles, on the survival rate of fibroblasts in the present invention;

[0038] Figure 8 The six groups of samples in the present invention are shown in Figure 1. The six groups of samples inhibit melanin synthesis and tyrosinase activity in B16 cells. The six groups of samples include: a. blank group; b. arbutin solution group; c. bakuchiol free drug group; d. bakuchiol micelle low concentration group; e. bakuchiol micelle medium concentration group; f. bakuchiol micelle high concentration group. The photos show the color state of melanocytes after treatment with different drug administration groups;

[0039] Fig. 9 It is a schematic diagram of the contents of hyaluronic acid, MMP-1, CoL-I and CoL-III in fibroblasts in the present invention;

[0040] Fig.10The six groups of samples in the present invention are fluorescence microscopy images showing the effects of six groups of samples on elastin in fibroblasts; wherein the six groups of samples include: a. blank group; b. arbutin solution group; c. bakuchiol free drug group; d. bakuchiol micelle low concentration group; e. bakuchiol micelle medium concentration group; f. bakuchiol micelle high concentration group;

[0041] Fig.11 It is a schematic diagram of the scratching effect of five different samples on Hacat cells in the present invention; wherein, Figure A is a schematic diagram of the scratching effect of five different samples on Hacat cells, and Figure B is a schematic diagram of the healing rate of five different samples on Hacat cells, and the five different samples include: a blank group, a free bakuchiol drug group, a high-concentration bakuchiol micelle group, a medium-concentration bakuchiol micelle group, and a low-concentration bakuchiol micelle group.

[0042] Fig.12 The six groups of samples in the present invention are HE stained paraffin-embedded sections of mice; wherein the six groups of samples include: a. blank group; b. normal saline group; c. bakuchiol free drug group; d. bakuchiol micelle low concentration group; e. bakuchiol micelle medium concentration group; f. bakuchiol micelle high concentration group.

[0043] Fig.13 It is a schematic diagram of the effects of six groups of different samples on the content of type I and type III collagen in the present invention; wherein, the six groups of different samples include: a blank group; a normal saline group; a bakuchiol free drug group; a bakuchiol micelle low concentration group; a bakuchiol micelle medium concentration group; and a bakuchiol micelle high concentration group.

[0044] Fig.14 It is a schematic diagram of the effect of six groups of different samples on the immunofluorescence of type I and type III collagen in the present invention; wherein, the six groups of different samples include: a blank group; a normal saline group; a free bakuchiol drug group; a low concentration group of bakuchiol micelles; a medium concentration group of bakuchiol micelles; and a high concentration group of bakuchiol micelles.

[0045] Fig.15 It is a schematic diagram of the effect of six groups of different samples on the immunofluorescence of type I and type III collagen in the present invention; wherein, the six groups of different samples include: a blank group; a normal saline group; a free bakuchiol drug group; a low concentration group of bakuchiol micelles; a medium concentration group of bakuchiol micelles; and a high concentration group of bakuchiol micelles. DETAILED DESCRIPTION

[0046] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used in the present application have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0047] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0048] The present invention is further described below in conjunction with specific examples, but the present invention is not limited to the following examples. The methods described are conventional methods unless otherwise specified.

[0049] Reagents used in the examples: DMEM medium (Gibico), double antibody (Gibico), fetal bovine serum (Gibico), 25 bottles / 75 bottles / 225 bottles / 96-well plate / 48-well plate / 6-well plate (Corning), CCK-8 (Dalian Meilun Biotechnology Co., Ltd.), levodopa (SLBW3857, Sigma), BCA kit (Dalian Meilun Biotechnology Co., Ltd.), stearic acid (SA, Shanghai Aladdin Biochemical Technology Co., Ltd.); Cistanche polysaccharide; 4-dimethylaminopyridine (DMAP, Shanghai Shanghai Aladdin Biochemical Technology Co., Ltd.); 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC·Cl, Shanghai Aladdin Biochemical Technology Co., Ltd.); anhydrous DMSO (Shanghai Aladdin Biochemical Technology Co., Ltd.); anhydrous ethanol (Shanghai Aladdin Biochemical Technology Co., Ltd.), potassium bromide (Shanghai Aladdin Biochemical Technology Co., Ltd.); bakuchiol (Chengdu Pufeide Biotechnology Co., Ltd.); 4,0,6-diamino-2-phenylindole (DAPI, Beijing Solarbio Technology Co., Ltd.).

[0050] Example 1

[0051] A method for preparing stearic acid-modified Cistanche deserticola polysaccharide comprises the following steps:

[0052] (1) Under nitrogen protection, 0.5 mmol of stearic acid (SA), 0.5 mmol of 4-dimethylaminopyridine (DMAP), and 0.5 mmol of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC·Cl) were dissolved in anhydrous dimethyl sulfoxide (DMSO), and the mixture was stirred at room temperature for 0.5 h to activate the carboxyl group, thereby obtaining a mixed solution;

[0053] (2) dissolving 400 mg of Cistanche polysaccharide in anhydrous dimethyl sulfoxide (DMSO), dissolving by ultrasonication to obtain a dissolved Cistanche polysaccharide solution, and dripping the mixed solution obtained in step (1) into the Cistanche polysaccharide solution, stirring at room temperature for 46 hours to react, and obtaining a reaction product;

[0054] (3) After the reaction is completed, 8 times the amount of anhydrous ethanol is added to the reaction product to precipitate overnight. The next day, the precipitate is collected and transferred to a dialysis bag with a molecular weight cutoff of 10,000. The water is changed in the morning and evening and dialyzed with distilled water for 46 hours to obtain a dialyzed product;

[0055] (4) freeze-drying the dialyzed product to obtain stearic acid-modified Cistanche deserticola polysaccharide.

[0056] Example 2

[0057] A method for preparing stearic acid-modified Cistanche deserticola polysaccharide comprises the following steps:

[0058] (1) Under nitrogen protection, 0.5 mmol of stearic acid (SA), 0.5 mmol of 4-dimethylaminopyridine (DMAP), and 0.5 mmol of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC·Cl) were dissolved in anhydrous dimethyl sulfoxide (DMSO), and the mixture was stirred at room temperature for 0.8 h to activate the carboxyl group, thereby obtaining a mixed solution;

[0059] (2) dissolving 400 mg of Cistanche polysaccharide in anhydrous dimethyl sulfoxide (DMSO), dissolving by ultrasonication to obtain a dissolved Cistanche polysaccharide solution, and dripping the mixed solution obtained in step (1) into the Cistanche polysaccharide solution, stirring at room temperature for 48 hours to react, and obtaining a reaction product;

[0060] (3) After the reaction is completed, 10 times the amount of anhydrous ethanol is added to the reaction product for precipitation overnight. The next day, the precipitate is collected and transferred to a dialysis bag with a molecular weight cutoff of 12,000. The water is changed in the morning and evening and dialyzed with distilled water for 48 hours to obtain the dialyzed product;

[0061] (4) freeze-drying the dialyzed product to obtain stearic acid-modified Cistanche deserticola polysaccharide.

[0062] Example 3

[0063] A method for preparing stearic acid-modified Cistanche deserticola polysaccharide comprises the following steps:

[0064] (1) Under nitrogen protection, 0.5 mmol of stearic acid (SA), 0.5 mmol of 4-dimethylaminopyridine (DMAP), and 0.5 mmol of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC·Cl) were dissolved in anhydrous dimethyl sulfoxide (DMSO), and the mixture was stirred at room temperature for 1 h to activate the carboxyl group to obtain a mixed solution;

[0065] (2) dissolving 400 mg of Cistanche polysaccharide in anhydrous dimethyl sulfoxide (DMSO), dissolving by ultrasonication to obtain a dissolved Cistanche polysaccharide solution, and dripping the mixed solution obtained in step (1) into the Cistanche polysaccharide solution, stirring at room temperature for 50 hours to react, and obtaining a reaction product;

[0066] (3) After the reaction is completed, 12 times the amount of anhydrous ethanol is added to the reaction product to precipitate overnight. The next day, the precipitate is collected and transferred to a dialysis bag with a molecular weight cutoff of 14,000. The water is changed in the morning and evening and dialyzed with distilled water for 50 hours to obtain a dialyzed product;

[0067] (4) freeze-drying the dialyzed product to obtain stearic acid-modified Cistanche deserticola polysaccharide.

[0068] Test example

[0069] 1.1 Characterization of stearic acid-modified Cistanche deserticola polysaccharide

[0070] The ratio of sample to KBr is 1:100. After grinding, the pressed KBr wafer is placed on a bracket and then placed on an infrared detector for detection. The infrared detection image obtained is as follows: Figure 1 As shown, Figure 1 The infrared characterization images of stearic acid, Cistanche deserticola polysaccharide and stearic acid-modified Cistanche deserticola polysaccharide in the present invention are shown in FIG. Figure 1 It can be seen that Cistanche polysaccharide has an obvious OH stretching vibration peak near 3500cm-1. In the infrared spectrum of stearic acid, there is an obvious carboxyl vibration absorption peak at around 1700cm-1, and there are stretching vibration absorption peaks belonging to the methylene group on the stearic acid chain at 2920cm-1 and 2850cm-1. Compared with Cistanche polysaccharide and stearic acid, in the infrared spectrum of stearic acid-modified Cistanche polysaccharide, in addition to retaining the OH stretching vibration peak of the sugar chain of Cistanche polysaccharide, new characteristic absorption peaks appear at 2920cm-1 and 2850cm-1, which are the characteristic peaks of the methylene group on the stearic acid chain. In addition, significant characteristic peaks of ester groups appear at 1735-1750cm-1. Since stearic acid is connected to the polysaccharide skeleton of Cistanche polysaccharide through ester bonds, the changes in these peaks indicate the successful synthesis of stearic acid-modified Cistanche polysaccharide.

[0071] 1.2 Characterization of the physicochemical properties of bakuchiol micelles encapsulated by stearic acid-modified Cistanche deserticola polysaccharide

[0072] 1.2.1 Preparation of Bakuchiol Micelle Encapsulated by Stearic Acid-Modified Cistanche Polysaccharide

[0073] 10 mg of the stearic acid-modified Cistanche polysaccharide prepared in Example 1 was dissolved in 5 mL of distilled water, and 1 mg of bakuchiol was dissolved in 1 mL of ethyl acetate. The two solutions were mixed, and ultrasonicated for 10 min (power: 500 W, single ultrasonic time 10 s, stop interval 5 s) with an ultrasonic cell crusher probe, moved to a round-bottom flask, and ethyl acetate was spin-dried (rotated until the solution volume was less than 5 mL) to obtain stearic acid-modified Cistanche polysaccharide-encapsulated bakuchiol micelles.

[0074] 1.2.2 Characterization of Bakuchiol Micelle Encapsulated by Stearic Acid-Modified Cistanche Polysaccharide

[0075] Particle size and zeta potential determination of micelles: The particle size, polydispersity index (PDI) and zeta potential value of the bakuchiol micelles encapsulated by stearic acid-modified Cistanche deserticola polysaccharide were evaluated using 500 nanometer particles and a zeta potential analyzer (n=3).

[0076] Encapsulation efficiency detection: The content of bakuchiol in the bakuchiol micelles before and after the column was determined by using a 1100 liquid chromatograph. 500 μL of bakuchiol micelles were accurately drawn into an EP tube, and an equal volume of methanol was added for ultrasonic demulsification. The chromatographic system used an Elite 1100 high performance liquid chromatograph, the chromatographic column used C18 (250 mm × 4.6 mm, 5 μm), the mobile phase of bakuchiol was methanol-water (75:25, V / V), the detection wavelength was 260 nm, the flow rate was 1 mL / min, the injection volume was 20 μL / time, and the column temperature was 30 ° C. The encapsulation efficiency (EE) calculation formula is: EE% = (W1 / W0) × 100%, where W1 represents the peak area of ​​the drug after the column, and W0 represents the peak area of ​​the drug before the column.

[0077] Figure 2 Figure 1 is a characterization diagram of the physicochemical properties of the stearic acid modified Cistanche polysaccharide-encapsulated Bakuchiol micelles in the present invention. Figure a is a Tyndall effect diagram, Figure b is a particle size diagram of the stearic acid modified Cistanche polysaccharide-encapsulated Bakuchiol micelles, and Figure c is a Zeta potential of the stearic acid modified Cistanche polysaccharide-encapsulated Bakuchiol micelles. Figure 2 It can be seen that the Tyndall effect is present, indicating that the prepared stearic acid-modified Cistanche polysaccharide-encapsulated bakuchiol micelles have good colloid performance. Potential and particle size characterization show that Zeta is around -6.5mV and the micelle particle size is 286.5nm.

[0078] 1.3 Investigation of the uptake of bakuchiol micelles (referred to as micelles) encapsulated by stearic acid-modified Cistanche deserticola polysaccharide in Hacat cells

[0079] 1.3.1 Fluorescence microscopy observation

[0080] Fluorescence microscopy was used to observe the cellular uptake of different preparations in Hacat cells.4 / well were inoculated in a 48-well plate. After incubation for 12 hours, 50 μL of free coumarin (coumarin concentration 3 μM) and different concentrations of coumarin micelles (coumarin concentrations were 1, 2, and 3 μM, respectively) were added respectively. After further culture for 2 hours, the culture medium was discarded, and the cells were washed with PBS to remove excess unabsorbed preparations. The cells were fixed with 4% paraformaldehyde for 15 minutes, and the cell nuclei were stained with DAPI in the dark for 10 minutes. The distribution of fluorescence in the cells was observed using a fluorescence microscope. Figure 3 is a diagram showing the distribution of fluorescence in cells in the present invention; Figure 3 It can be seen that the fluorescence intensity of the micelle group is enhanced compared with free coumarin, indicating that the micelle carrier enhances the uptake of drugs by Hacat cells. As the concentration of coumarin increases, the fluorescence intensity further increases. These results show that amphiphilic micelles improve the bioavailability of drugs.

[0081] 1.3.2 Flow cytometer observation

[0082] Flow cytometry was used to detect the cellular uptake of different preparations in Hacat cells. The specific method was the same as above. After adding different preparations and continuing to culture for 2 hours, the culture medium was discarded, PBS was washed, the cells were trypsinized, centrifuged and washed, and pre-cooled PBS was added to resuspend the cells. The fluorescence intensity of the probe bound to the cells was quantitatively analyzed by flow cytometry. Figure 4 : is an analysis diagram of the quantitative analysis of the fluorescence intensity of the probe bound to the cells by using a flow cytometer in the present invention; Figure 4 As can be seen in the figure: The results of flow cytometry also show that the fluorescence intensity of the micelle-encapsulated coumarin group is higher than that of the free coumarin group. The results of flow cytometry are similar to those of fluorescence microscopy, both of which illustrate the beneficial effects of amphiphilic micelles.

[0083] 1.4 Investigation of micelle retention in the skin

[0084] Using the vertical Franz diffusion cell method, pig skin with similar permeability to human skin was selected as a skin model, and the retention of micelles in the skin was investigated through in vitro transdermal experiments. The stratum corneum of the pig skin was fixed in the middle of the Franz diffusion cell, with the dermis facing the supply cell and the stratum corneum facing the receiving cell. The transdermal area was about 3cm 2, add 8.5mL of physiological saline (meeting the sink condition) to the receiving pool with a pipette, keep the water bath temperature at (36±0.5)℃, and add it to the supply chamber. Take 1mL of different preparations and add them to the supply pool for transdermal experiments. After 2h, take out the pig skin, cut it into appropriate size, embed it with OCT frozen section embedding agent to make specimens, and store it in a refrigerator at -20℃. Place the specimen on the support table and slice it with a Leica cryostat. The slice thickness is 10μm. DAPI is used to stain the cell nucleus in the dark for 10min. The retention of micelles in the skin is observed with a fluorescence microscope and photographed. Figure 5 is a fluorescence microscope picture of the retention of micelles in the skin in the present invention; Figure 5 It can be seen that the fluorescence intensity of coumarin is used to represent the effect of drug retention in the skin. The stronger the fluorescence intensity, the better the retention effect. The fluorescence intensity corresponding to the low-concentration coumarin micelle group is higher than the fluorescence intensity of the high-concentration free coumarin, which means that the micelle encapsulation significantly improves the retention of the drug in the skin. Similarly, as the drug concentration increases, the retention of micelle-encapsulated drugs in the skin is further improved, and the results are consistent with the above-mentioned cell uptake experimental results.

[0085] 1.5 Evaluation of cytotoxic activity

[0086] 1.5.1 Effect of the test substance on the survival rate of mouse melanoma cells (B16 cells)

[0087] B16 cells in good logarithmic growth phase were cultured at 2×10 4 The cells were inoculated at a density of 100 / mL in a 96-well culture plate and incubated at 37°C with 5% CO 2 Incubate overnight in the environment. Add different concentrations of sample solutions, including: free bakuchiol, bakuchiol micelles and blank micelles; make seven different concentrations of each sample, and make five parallels for each concentration. The cell control group is not treated and cultured for 48 hours. Add 10μL CCK8 solution to each well, culture for 2.5 hours, and measure the absorbance of each well at 450nm. Cell survival rate = (OD value of the measured well - OD value of the blank control) / (OD value of the cell control group - OD value of the blank control) * 100%. Figure 6 This is a graph showing the effects of the three samples of bakuchiol, bakuchiol micelles and blank micelles on the survival rate of mouse melanoma cells (B16 cells) in the present invention.

[0088] 1.5.2 Effect of the test substance on the survival rate of fibroblasts

[0089] Fibroblasts in good logarithmic growth phase were cultured at 1×10 4The cells were inoculated at a density of 100 μL / mL in a 96-well culture plate and cultured overnight at 37°C in a 5% CO2 environment. Sample solutions of different concentrations were added, including free bakuchiol, micelles of bakuchiol and blank micelles; seven different concentrations were made for each sample, and five parallels were made for each concentration. The cell control group was not treated and cultured for 48 hours. 10 μL CCK8 solution was added to each well, and after culturing for 2.5 hours, the absorbance of each well was measured at 450 nm. Cell survival rate = (OD value of the measured well - OD value of the blank control) / (OD value of the cell control group - OD value of the blank control) * 100%. Figure 7 This is a graph showing the effects of the three samples of bakuchiol, bakuchiol micelles and blank micelles on the survival rate of fibroblasts in the present invention.

[0090] from Figure 6 and Figure 7 It can be seen that the effects of free bakuchiol, bakuchiol micelles and blank micelles on the survival rates of B16 cells and fibroblasts are basically the same. When the concentrations of free bakuchiol and bakuchiol micelles are 100 μg / mL, there is no cell inhibition rate on both types of cells. In subsequent cell tests, the concentration of free bakuchiol was set to 100 μg / mL, and the concentrations of bakuchiol micelles were set to 100 μg / mL, 50 μg / mL and 10 μg / mL.

[0091] 1.6 Whitening activity efficacy test

[0092] 1.6.1 Melanin synthesis inhibition experiment

[0093] B16 cells in good logarithmic growth phase were taken at 1×10 5 The cells were inoculated at a density of 100 μg / mL in a 6-well plate and cultured overnight in an incubator at 37°C and 5% CO2. The culture medium was replaced and samples of different concentrations were added, including: a. blank group; b. arbutin solution group; c. bakuchiol free drug group; d. bakuchiol micelle low concentration group; e. bakuchiol micelle medium concentration group; f. bakuchiol micelle high concentration group. The negative control group was not treated. After 48 hours of culture, the culture medium was discarded, 250 μL of trypsin was added after washing with PBS for 4 minutes, 1 mL of culture medium was added to terminate the digestion, the cells were centrifuged at 1500 rpm for 3 minutes, the supernatant was discarded, 0.5 ml of 1 M sodium hydroxide solution containing 10% DMSO was added, ultrasonic dispersion was performed for 3 minutes, 20 μL of the above solution was taken to determine the BCA kit, and the remaining solution was water bathed at 80°C for 30 minutes; the cells were shaken and mixed, centrifuged at 10000 rpm for 2 minutes, 200 μL of the supernatant was aspirated to a 96-well plate, and the absorbance value was read at 405 nm. Melanin content change = (OD value of the assay well - OD value of the blank control) / (OD value of the cell control group - OD value of the blank control). Each group of components was repeated 5 samples, and the melanin content change rate required BCA content correction.

[0094] 1.6.2 Inhibition of tyrosinase activity in B16 cells

[0095] B16 cells in good logarithmic growth phase were taken at 1×10 5 The cells were inoculated at a density of 100 / mL in a 6-well plate and cultured overnight in an incubator at 37°C and 5% CO2. The culture medium was replaced and samples of different concentrations were added, including: a. blank group; b. arbutin solution group; c. bakuchiol free drug group; d. bakuchiol micelle low concentration group; e. bakuchiol micelle medium concentration group; f. bakuchiol micelle high concentration group; the negative control group was not treated. After 48 hours of culture, the culture medium was discarded, washed with PBS and added with 250 μL trypsin digestion for 4 minutes, add 1mL culture medium to stop digestion, centrifuge at 1500rmp for 3 minutes, discard the supernatant, add 300μL of 1% TritonX-100 and shake and sonicate for 5 minutes, take 20μL of the above solution to measure the BCA kit, take 100μL of the remaining solution to a 96-well plate, preheat at 37℃ for 5 minutes, then add 100μL of 0.25% L-dopa solution, incubate at 37℃ for 1 hour, and read the absorbance at 490nm. Change in tyrosinase activity = (OD value of the assay well - OD value of the blank control) / (OD value of the cell control group - OD value of the blank control). Repeat 5 samples for each group of components, and the change in tyrosinase activity needs to be corrected by BCA content.

[0096] Figure 8 The six groups of samples in the present invention are shown in Figure 2 for the inhibition of melanin synthesis and tyrosinase activity in B16 cells; the six groups of samples include: a. blank group; b. arbutin solution group; c. bakuchiol free drug group; d. bakuchiol micelle low concentration group; e. bakuchiol micelle medium concentration group; f. bakuchiol micelle high concentration group; the photos show that there are differences in the color of melanocytes treated with different drug administration groups. The color of melanocytes treated with the positive drug arbutin is the lightest, and the color of melanocytes corresponding to free bakuchiol and bakuchiol micelles is also lighter than that of the control group. In addition, as the drug concentration of the micelle group increases, the color of melanocytes gradually becomes lighter. Figure 8 It can be seen that the melanin inhibition rate and tyrosinase inhibition rate of bakuchiol micelles increase with the increase of bakuchiol micelle concentration. Moreover, the inhibitory ability of bakuchiol micelles on melanin and tyrosinase is concentration-dependent. Tyrosinase is a key enzyme in melanin synthesis, and bakuchiol micelles can further reduce the formation of melanin by blocking tyrosinase activity.

[0097] 1.7 Anti-aging efficacy testing

[0098] 1.7.1 Determination of hyaluronic acid, MMP-1, CoL-I and CoL-III in fibroblasts

[0099] Experimental plan: Count the human fibroblasts in good logarithmic growth phase and inoculate them into 6 cm culture dishes, with the number of cells per dish controlled at 2×10 5 Cells were cultured overnight at 37°C and 5% CO2, the culture medium was discarded, and samples of different concentrations were added to act on the cells for 6 hours (serum-free DMEM culture medium was added to the model group and the blank control group), a small amount of PBS (Ph=7.4) was added to just cover the cells, and the cells were stimulated with UVA, with a UV radiation dose of 7J / cm 2 , irradiation time 2h, blank group without irradiation. Each sample was tested three times (each parallel sample was tested three times), taken out, placed on ice, washed twice with PBS; 500μL lysis buffer was added to lyse the cells, centrifuged at 12000rmp at 4℃ for 5min, and the supernatant was obtained to obtain the cell lysis supernatant. The content of hyaluronic acid, MMP-1, CoL-I and CoL-III in the supernatant was determined according to the ELISA kit detection steps, and the results needed to be corrected with BCA content. Fig. 9 Schematic diagram of the contents of hyaluronic acid, MMP-1, CoL-I and CoL-III in fibroblasts of the present invention; Fig. 9 It can be seen that the contents of hyaluronic acid, collagen I (COL-I) and collagen III (COL-III) in aged skin cells decrease, while the content of matrix metalloproteinase (MMP-1) increases. The results show that bakuchiol micelles can increase the contents of hyaluronic acid, collagen I (COL-I) and collagen III (COL-III) in aged cells, reduce the content of MMP-1, and high concentrations, i.e. 100 μg / mL of bakuchiol micelles, can achieve an effect close to that of the positive drug vitamin C. These results show that bakuchiol micelles can improve the aging of skin cells.

[0100] 1.7.2 Determination of elastin in fibroblasts

[0101] Human fibroblasts in good logarithmic growth phase were counted and inoculated into 6-well plates, and the number of cells per well was controlled to be 1×106 cells. Cultured overnight at 37°C, 5% CO2, discarded the culture medium, and added different samples of the same (optimal) concentration, where the samples included: a. blank group; b. arbutin solution group; c. bakuchiol free drug group; d. bakuchiol micelle low concentration group; e. bakuchiol micelle medium concentration group; f. bakuchiol micelle high concentration group; act on the cells for 6 hours (serum-free DMEM culture medium was added to the model group and the blank control group), add a small amount of PBS (Ph=7.4) to just cover the cells, and stimulate the cells with UVA, with a UV radiation dose of 7J / cm 2, irradiation time 2 hours, blank group was not irradiated. After removing the culture medium and washing once with sterile PBS, 1000 μL of 4% paraformaldehyde phosphate buffer (pH 7.5) was added and fixed at 4°C for 10 minutes. After washing once with sterile PBS, 10% goat serum-PBS solution was added, and the mixture was allowed to stand at room temperature for 30 minutes. As a primary antibody, anti-elastin antibody (11C1.3, mouse; CLOUD-CLONE CORP) was diluted 200 times with 0.1% BSA-PBS, and 200 μL was added to the coverslip site and allowed to stand at room temperature for 1 hour and 30 minutes. After washing 3 times with PBS, AlexaFluor 488 goat anti-mouse IgG was diluted 1000 times with 0.1% BSA, 200 μL was added to each sample, and then allowed to stand at room temperature for 40 minutes. DAPI (nuclear stain) was diluted 2000 times with PBS, and then 500 μL was added. This was left at room temperature for about 10 minutes, washed thoroughly with PBS, 1000 μL of PBS was added, and the cells were observed with a fluorescence microscope. Fig.10 The fluorescence microscope images of the effects of six different samples on elastin in fibroblasts in the present invention are shown in FIG. 6 ; wherein the six different samples include: a. blank group; b. arbutin solution group; c. bakuchiol free drug group; d. bakuchiol micelle low concentration group; e. bakuchiol micelle medium concentration group; f. bakuchiol micelle high concentration group; Fig.10 It can be seen that both the free bakuchiol group and the micelle group can increase the content of elastin, especially the micelle group. Moreover, with the increase of micelle concentration, the improvement effect of micelle group on elastin content is more significant, showing concentration dependence.

[0102] 1.8 Wound repair experiment

[0103] 1.8.1 Scratch test

[0104] 1×10 5 Hacat cells were inoculated in a 12-well plate at a cell concentration of 100 / well. When the number of cells in the culture plate reached 70%, a horizontal line was gently drawn on the bottom of the culture plate with a micropipette tip. The broken Hacat cells were rinsed twice with PBS buffer and then supplemented with fresh DMEM culture medium. Finally, each group of preparations was given, and the final concentration of bakuchiol free drug was 100μg / mL; the final concentration of bakuchiol micelles was 100μg / mL (bakuchiol micelle high concentration group), 50μg / mL (bakuchiol micelle medium concentration group) and 10μg / mL (bakuchiol micelle low concentration group). An inverted microscope was used to take pictures at 0, 12 and 24h and observe the difference in scratch width between the groups. Fig.11Schematic diagram of scratching Hacat cells with five different samples in the present invention; wherein, Figure A is a schematic diagram of the scratch experiment, and Figure B is a statistical diagram of the healing rate, and the five different groups of samples include: a blank group, a free bakuchiol drug group, a high-concentration bakuchiol micelle group, a medium-concentration bakuchiol micelle group, and a low-concentration bakuchiol micelle group; from Fig.11 It can be seen that both the free bakuchiol group and the micelle group can promote the migration of Hacat cells, especially the medium and high concentrations of bakuchiol micelles. The healing rates of high concentrations of bakuchiol micelles at 12h and 24h were 42% and 50%, respectively. This shows that the encapsulation of micelles improves the bioavailability of bakuchiol and enhances its effect in promoting cell migration.

[0105] 1.9 In vivo anti-aging experiments

[0106] The photoaging model was established by irradiating BALB / c Nude mice with ultraviolet UVB, and the initial dose was set at 36 mJ / m 2 , and then increased to 54, 72, 108, 144, 162, 180 and 198 mJ / m 2 , 1 week apart, and finally 216 mJ / cm at the 9th and 10th weeks 2 The irradiation frequency was set to 3 times per week (30 times / 10 weeks). The BALB / c Nude mice in the blank control group were not irradiated; after 10 weeks of irradiation, the aging model mice were randomly divided into 5 groups, 3 mice in each group, and applied with normal saline, free bakuchiol (100μg / ml), high-concentration bakuchiol micelle group (100μg / ml), medium-concentration bakuchiol micelle group (50μg / ml) and low-concentration bakuchiol micelle group (10μg / ml).

[0107] 1.9.1 Histological observation

[0108] Routine paraffin-embedded sections were performed for HE staining.

[0109] Fig.13 The six groups of samples in the present invention are HE stained paraffin-embedded sections of mice; wherein the six groups of samples include: a. blank group; b. saline group; c. bakuchiol free drug group; d. bakuchiol micelle low concentration group; e. bakuchiol micelle medium concentration group; f. bakuchiol micelle high concentration group; from Fig.13 It can be seen that after skin aging, the epidermal thickness of the model group increased compared with the normal group, and both free bakuchiol and bakuchiol micelles can reduce the epidermal thickness. Among them, as the concentration of bakuchiol micelles increases, the more obvious the reduction in epidermal thickness, the better the effect of improving aging.

[0110] 1.9.2 ELISA kit to determine the content of type I and type III collagen

[0111] Ice-cold tissue protein lysate was added to the skin sample at a ratio of sample weight: tissue protein lysate = 1:4 (g / ml), wherein the samples included: a. blank group; b. saline group; c. bakuchiol free drug group (100μg / ml); d. bakuchiol micelle low concentration group (10μg / ml); e. bakuchiol micelle medium concentration group (50μg / ml); f. bakuchiol micelle high concentration group (100μg / ml); then the skin was homogenized, and the homogenate was centrifuged at 4℃, 13500rpm / min for 10min. The supernatant after centrifugation was used to determine the content of type I and type III collagen in the sample using an ELISA kit. The determination of collagen I and III in the homogenate was in accordance with the instructions of the kit, and the final result required BCA content correction. Fig.14 Schematic diagram of the effect of six different samples on the content of type I and type III collagen in the present invention; wherein the six different samples include: blank group; saline group; bakuchiol free drug group; bakuchiol micelle low concentration group; bakuchiol micelle medium concentration group; bakuchiol micelle high concentration group; Fig.14 It can be seen that the contents of COL-I and COL-III in the skin of the model group were lower than those in the skin of normal mice, and both free bakuchiol and bakuchiol micelles increased the contents of COL-I and COL-III in the skin, especially the bakuchiol micelles, and the improvement effect of bakuchiol micelles increased with the increase of concentration.

[0112] 1.9.3 Immunofluorescence detection of type I and type III collagen content

[0113] After conventional dewaxing and hydration, the paraffin-embedded sections were placed in a citric acid antigen repair solution for microwave heating for 15 minutes, and washed 3 times with PBS; incubated with 3% H2O2 for 5 minutes, and washed 3 times with PBS; blocked with 5% BSA solution for 30 minutes, and Col-I antibody and Col-III antibody (1:500) dilutions were added to the sections, incubated at 4°C overnight, and the next day, the antibody dilutions were discarded, and washed 3 times with PBS; fluorescent secondary antibody (1:500) dilutions were added, incubated at room temperature in the dark for 1 hour, and washed 3 times with PBS; DAPI was used to stain the nucleus for 10 minutes, washed 3 times with PBS, and sealed with aqueous sealing solution. The positive expression of Col-I was observed under an inverted fluorescence microscope. Fig.15 Schematic diagram of the effect of six different samples on type I and type III collagen immunofluorescence in the present invention; wherein the six different samples include: blank group; saline group; bakuchiol free drug group; bakuchiol micelle low concentration group; bakuchiol micelle medium concentration group; bakuchiol micelle high concentration group; Fig.15It can be seen that the fluorescence intensity of COL-I and COL-III in the skin of the model group is lower than that of the normal mouse skin. Both free bakuchiol and bakuchiol micelles increase the corresponding fluorescence intensity of COL-I and COL-III in the skin of the model mice, especially the bakuchiol micelles. Moreover, the improvement effect of bakuchiol micelles increases with the increase of concentration. This is consistent with the above BCA experimental results.

[0114] In summary, the stearic acid-modified Cistanche polysaccharide-encapsulated bakuchiol micelles prepared in the present invention have good whitening, anti-aging and wound repair effects, and can be used to prepare medicines or cosmetics with whitening, anti-aging and wound repair effects.

[0115] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing bakuchiol micelles entrapped by stearic acid-modified Cistanche deserticola polysaccharide, characterized in that: The following steps are involved: (1) Under nitrogen protection, stearic acid, 4-dimethylaminopyridine and 1-ethyl-(3-dimethylaminopropyl)carbodiimide were dissolved in anhydrous dimethyl sulfoxide, and the carboxyl groups were activated by stirring at room temperature to obtain a mixed solution; (2) dissolving Cistanche polysaccharide in anhydrous dimethyl sulfoxide to obtain a dissolved Cistanche polysaccharide solution, adding the mixed solution obtained in step (1) dropwise to the Cistanche polysaccharide solution, stirring at room temperature for a period of time to react, and obtaining a reaction product; (3) After the reaction is completed, anhydrous ethanol is added to the reaction product for alcohol precipitation, and after the alcohol precipitation is completed, the precipitate is collected and dialyzed to obtain a dialyzed product; (4) freeze-drying the dialyzed product to obtain stearic acid-modified Cistanche deserticola polysaccharide; (5) Dissolving the stearic acid-modified Cistanche polysaccharide in water to obtain solution 1, dissolving bakuchiol in ethyl acetate to obtain solution 2, mixing solution 1 and solution 2, and removing the ethyl acetate after ultrasonic treatment to obtain stearic acid-modified Cistanche polysaccharide-encapsulated bakuchiol micelles.

2. The method for preparing the stearic acid-modified Cistanche polysaccharide-encapsulated bakuchiol micelles according to claim 1, characterized in that: In step (1), the molar ratio of stearic acid, 4-dimethylaminopyridine and 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 0.01-1.0: 0.01-1.0: 0.01-1.

0.

3. The method for preparing the stearic acid-modified Cistanche polysaccharide-encapsulated bakuchiol micelles according to claim 1, characterized in that: The stirring time at room temperature in step (1) is 0.5 to 1 h.

4. The method for preparing the stearic acid-modified Cistanche polysaccharide-encapsulated bakuchiol micelles according to claim 1, characterized in that: The stirring time at room temperature in step (2) is 46 to 50 h.

5. The method for preparing the stearic acid-modified Cistanche polysaccharide-encapsulated bakuchiol micelles according to claim 1, characterized in that: The alcohol precipitation step in step (3) is as follows: add 8 to 12 times the amount of anhydrous ethanol to the reaction product for alcohol precipitation overnight, collect the dialyzed product the next day and transfer it to a dialysis bag, change the water in the morning and evening, and dialyze with distilled water for 46 to 50 hours.

6. The method for preparing the stearic acid-modified Cistanche polysaccharide-entrapped bakuchiol micelles according to claim 1, characterized in that: The molecular weight cut-off of the dialysis bag is 10,000-14,000.

7. Bakuchiol micelles entrapped by stearic acid-modified Cistanche polysaccharide prepared by the method for preparing bakuchiol micelles entrapped by stearic acid-modified Cistanche polysaccharide according to any one of claims 1 to 6.

8. A method for preparing the stearic acid-modified Cistanche polysaccharide-encapsulated bakuchiol micelles according to any one of claims 1 to 6 and / or use of the stearic acid-modified Cistanche polysaccharide-encapsulated bakuchiol micelles according to claim 7 in the preparation of medicines or cosmetics with whitening effects.

9. A method for preparing the stearic acid-modified Cistanche polysaccharide-encapsulated bakuchiol micelles according to any one of claims 1 to 6 and / or use of the stearic acid-modified Cistanche polysaccharide-encapsulated bakuchiol micelles according to claim 7 in the preparation of medicines or cosmetics with anti-aging effects.

10. The method for preparing the stearic acid-modified Cistanche polysaccharide-encapsulated bakuchiol micelles according to any one of claims 1 to 6 and / or the use of the stearic acid-modified Cistanche polysaccharide-encapsulated bakuchiol micelles according to claim 7 in the preparation of medicines or cosmetics with wound repairing efficacy.

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