Self-assembled supramolecular raw material of hydroxypropyl-β-cyclodextrin and epigallocatechin gallate betaine, preparation method and application thereof

Abstract

The present invention relates to the technical fields of medicine, food and daily chemicals, and in particular to two supramolecular eutectics to better solve the application problems of EGCG. The first is to form a supramolecular EGCG betaine eutectic with EGCG and betaine through supramolecular technology, and to synthesize a supramolecular EGCG betaine eutectic by supramolecular modification reaction using EGCG and betaine as precursors, which can improve the stability of EGCG to a certain extent, and the two can be used in combination to play a synergistic role. The second is to form a supramolecular raw material with a supramolecular EGCG betaine eutectic and cyclodextrin through host-guest self-assembly, and to obtain a supramolecular raw material by host-guest self-assembly of hydroxypropyl-β-cyclodextrin and supramolecular EGCG betaine. The supramolecular raw material obtained by the method can solve the stability problem of EGCG, improve its bioavailability, and have good anti-oxidation, whitening, anti-inflammatory, oil control and other effects, and has good application prospects.

Description

Technical Field

[0001] The invention relates to the technical fields of medicine, food and daily chemicals, and in particular to a self-assembled supramolecular raw material of hydroxypropyl-beta-cyclodextrin and epigallocatechin gallate betaine, and a preparation method and application thereof. Background Art

[0002] Tea polyphenols are the main components that determine the color, aroma, taste and efficacy of tea, accounting for 20% to 30% of the dry weight of tea. Tea polyphenols are divided into four major categories according to their main chemical components: catechins, flavonoids, anthocyanins, and phenolic acids. Among them, catechins have the highest content, accounting for 60% to 80% of tea polyphenols. Catechins are mainly epicatechin (EC), epigallocatechin (EGC), epicatechin gallate (ECG) and epigallocatechin gallate (EGCG). Among the four main catechins, epigallocatechin gallate has the strongest antioxidant activity and is also known as the "UV filter". Epigallocatechin gallate contains a large number of hydrophilic groups, which can easily absorb moisture from the air, inhibit the activity of hyaluronidase, and reduce the loss of skin moisture; epigallocatechin gallate can effectively scavenge free radicals and regulate the activity of enzymes related to photoaging, resist pollution, and prevent and treat skin aging and wrinkles. However, the special chemical structure of epigallocatechin gallate makes it easy to undergo oxidation reaction when it encounters external environmental factors such as light, high temperature, and alkalinity, thereby causing epigallocatechin gallate to deteriorate, causing it to lose its original efficacy or reduce its bioavailability, so its application needs to be developed and improved. Therefore, a new type of molecular raw material is urgently needed to solve the application problem of epigallocatechin gallate being unstable when exposed to light and high temperature and easily oxidized, resulting in low bioavailability.

[0003] Betaine is an alkaloid that is mainly found in the molasses of beet sugar, hence the name. Betaine is commonly found in animals and plants, and is an intermediate product of animal metabolism. It plays a very important role in the metabolism of nutrients. Betaine is a very important osmotic regulator, and is very important for plants to enhance stress resistance, such as salt and alkali resistance and drought resistance. At the same time, betaine can play a good cleansing role without harming the skin. It is very safe and is often used in cleansing skin care products. It is safe and effective, and has the effect of reducing skin irritation.

[0004] Epigallocatechin gallate has antioxidant, whitening, soothing, and repairing effects, and betaine has moisturizing and repairing effects, but the combination of the two to exert a synergistic effect and improve the stability and bioavailability of epigallocatechin gallate has not been reported. In addition, in recent years, cocrystal formation has become a feasible strategy to improve the stability and bioavailability of active pharmaceutical ingredients. By co-crystallizing the active ingredient with a coformer, a new solid form with unique properties compared to the active ingredient is formed, thereby improving the pharmaceutical preparation and improving its stability and bioavailability. However, cocrystal formation is unpredictable and is not always possible in fact. In addition, there is no way to predict the properties of the specific cocrystal before the compound is formed. Finding suitable coformers and appropriate conditions to obtain specific cocrystals requires researchers to spend more creative labor.

[0005] Cyclodextrin (CD) is a general term for a series of cyclic oligosaccharides produced by amylose under the action of cyclodextrin glucosyltransferase produced by Bacillus. Among them, the molecules containing 6, 7, and 8 glucose units are studied more and have important practical significance, which are called α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin, respectively. Cyclodextrin molecules have a slightly conical hollow cylindrical three-dimensional ring structure. In its hollow structure, the upper end (larger opening end) of the outside is composed of secondary hydroxyl groups of C2 and C3, and the lower end (smaller opening end) is composed of primary hydroxyl groups of C6, which are hydrophilic. The cavity forms a hydrophobic area due to the shielding effect of C-H bonds. Because the outer edge of cyclodextrin is hydrophilic and the inner cavity is hydrophobic, it can provide a hydrophobic binding site like an enzyme, as a main body to envelop various appropriate objects, such as organic molecules, inorganic ions, and gas molecules. Its hydrophobic inner cavity and hydrophilic outer cavity enable it to form inclusion complexes and molecular assembly systems with many organic and inorganic molecules based on van der Waals forces, hydrophobic interaction forces, and matching effects between host and guest molecules. Hydroxypropyl-β-cyclodextrin is the introduction of hydroxypropyl to break the intramolecular cyclic hydrogen bonds of β-cyclodextrin, while maintaining the cyclodextrin cavity and overcoming the main disadvantage of β-cyclodextrin's poor water solubility. Therefore, hydroxypropyl-β-cyclodextrin is one of the most intensively studied and widely used cyclodextrin derivatives, mainly used in the food, medicine, and cosmetics industries.

[0006] Therefore, there is an urgent need for a new supramolecular raw material to fully solve the application problem of low bioavailability of epigallocatechin gallate, which is unstable when exposed to light and high temperature and easily oxidized. Summary of the invention

[0007] In view of the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide a hydroxypropyl-β-cyclodextrin and supramolecular epigallocatechin gallate betaine self-assembled supramolecular raw material and its preparation method and application, aiming to solve the application problem of low bioavailability of epigallocatechin gallate due to its instability under light and high temperature and easy oxidation.

[0008] Supramolecular cocrystals are supramolecular structures formed by the mutual recognition between molecules of different types through intermolecular forces (including hydrogen bonds, van der Waals forces, π-π stacking and electrostatic interactions, etc.). Cocrystals are mainly formed in the mode of API+CCF, and CCF can be single or multiple. APIs usually contain various functional groups and have different active substances (for example: acids, bases, salts, non-ionic compound molecules, etc.); CCFs are connected in the same crystal lattice by non-covalent bonds such as hydrogen bonds, and are usually called ligands. An API can usually form cocrystals with multiple single ligands, and can also form multifunctional ions or cocrystal polymers with regular and stable structures through intermolecular self-assembly with multiple ligands.

[0009] The invention uses epigallocatechin gallate and betaine to prepare a supramolecular epigallocatechin gallate betaine with high efficacy in resisting photoaging through supramolecular eutectic technology, and then uses hydroxypropyl-β-cyclodextrin as a main body to self-assemble the supramolecular epigallocatechin gallate betaine eutectic as a guest substance under weak hydrogen bond interaction, thereby perfectly solving the problem of epigallocatechin gallate stability, and at the same time, the supramolecular raw materials after double encapsulation can better exert the efficacy of epigallocatechin gallate and betaine, thereby laying a foundation for wide application in medicine, food, daily chemicals and the like.

[0010] The technical solution of the present invention is as follows:

[0011] The first aspect of the present invention provides a method for preparing a supramolecular raw material for self-assembly of hydroxypropyl-β-cyclodextrin and supramolecular epigallocatechin gallate betaine, comprising the following steps:

[0012] S1. Add a solvent and epigallocatechin gallate and betaine in a molar ratio of 1:1-1:3 into an ultrasonic reactor, apply ultrasonic energy at a first reaction stirring speed of 400-800 rpm and a first reaction temperature of 30-50° C., an ultrasonic power of 1000-2000 w, an ultrasonic frequency of 10-25 kHz, and continue the reaction for 1-6 hours to form a suspension of epigallocatechin gallate and betaine;

[0013] S2, transferring the suspension to an evaporation crystallization reaction tank, and cooling and crystallizing at a second reaction temperature of -10 to 5°C to obtain supramolecular epigallocatechin gallate betaine eutectic;

[0014] S3, adding a solvent and a hydroxypropyl-β-cyclodextrin and supramolecular epigallocatechin gallate betaine cocrystal in a molar ratio of 1:1 to 1:5 into a reactor, stirring the second reaction at a second reaction speed of 600 to 1000 rpm and a third reaction temperature of 25 to 45° C. for a second reaction time of 6 to 12 hours to form a suspension of hydroxypropyl-β-cyclodextrin and supramolecular epigallocatechin gallate betaine;

[0015] S4, transferring the suspension from the reactor to a high-pressure homogenizer, homogenizing the solution by high pressure, wherein the pressure of the homogenizing valve is 100-300 MPa, and performing dialysis treatment after homogenization to form a dispersed stock solution of hydroxypropyl-β-cyclodextrin and supramolecular epigallocatechin gallate betaine;

[0016] S5. Transfer the dispersed stock solution to a spray dryer, set the feed temperature and spray speed for spray drying, and obtain a self-assembled supramolecular raw material of hydroxypropyl-β-cyclodextrin and supramolecular epigallocatechin gallate betaine.

[0017] Furthermore, the solvent in step S1 is anhydrous ethanol; the solvent in step S3 is distilled water.

[0018] Furthermore, in the step S4, the dialysis treatment uses a cellulose dialysis bag, the molecular weight of the cellulose dialysis bag is 500-1000; the dialysis treatment time is 2-4 hours, and the number of dialysis is 1-3 times.

[0019] Furthermore, the feed temperature of the spray drying process in step S5 is set to 150-180° C., and the spraying speed of the spray drying process is 8-10 mL / min.

[0020] The second aspect of the present invention provides a self-assembled supramolecular raw material of hydroxypropyl-β-cyclodextrin and epigallocatechin gallate betaine prepared according to the above preparation method.

[0021] Furthermore, the supramolecular epigallocatechin gallate betaine cocrystal in the self-assembled supramolecular raw material is encapsulated in the cavity of hydroxypropyl-β-cyclodextrin; and the molar ratio of hydroxypropyl-β-cyclodextrin to the supramolecular epigallocatechin gallate and betaine cocrystal is 1:1-1:5.

[0022] Furthermore, the supramolecular epigallocatechin gallate betaine cocrystal is a supramolecular cocrystal formed by weak interactions such as epigallocatechin gallate and betaine through hydrogen bonds and van der Waals interactions. The structural formula of the supramolecular epigallocatechin gallate betaine is:

[0023] ;

[0024] The molar ratio of epigallocatechin gallate to betaine in the supramolecular eutectic is 1:1-1:3.

[0025] Furthermore, in the X-ray diffraction pattern of the supramolecular epigallocatechin gallate betaine cocrystal, at a 2θ angle of 5.7±0.2 0 , 8.9±0.2 0 , 10.8±0.2 0 , 21.9±0.2 0 and 26.3±0.2 0 , or at the crystal plane spacing d of 15.63±0.2Å, 9.91±0.2Å, 8.18±0.2Å, 4.06±0.2Å, 3.57±0.2Å, and 3.39±0.2Å, characteristic peaks with relative intensities of 4.9%, 8.2%, 4.7%, 44.4%, 100.0%, and 83.0% appear, respectively.

[0026] The third aspect of the present invention provides the use of the above-mentioned hydroxypropyl-β-cyclodextrin and epigallocatechin gallate betaine self-assembled supramolecular raw material in the fields of medicine, food and daily chemicals.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The present invention uses hydroxypropyl-β-cyclodextrin as the main body, and under weak interactions such as hydrogen bonds and van der Waals interactions, uses the supramolecular eutectic formed by epigallocatechin gallate and betaine as the guest substance for host-guest self-assembly, which fully solves the problem that epigallocatechin gallate is easily oxidized and discolored. The epigallocatechin gallate and betaine of the present invention are used in combination, which can play a synergistic role, promote the penetration of active ingredients, improve bioavailability, and significantly reduce their irritation, avoid the risk of causing damage to the skin, and achieve gentle and efficient drug delivery. In addition, the present invention also has the advantages of supramolecular raw materials after double wrapping, so as to better play the effects of epigallocatechin gallate and betaine, and lay a foundation for their wide application in medicine, food, daily chemicals, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The method of the present invention and its beneficial effects are described in detail below in conjunction with the accompanying drawings and specific implementation methods.

[0030] Figure 1 The present invention is a schematic diagram of the process flow of the method for preparing the supramolecular raw material of self-assembly of hydroxypropyl-β-cyclodextrin and supramolecular epigallocatechin gallate betaine.

[0031] Figure 2 This is the molecular distribution diagram of the components of the supramolecular epigallocatechin gallate betaine cocrystal.

[0032] Figure 3 Schematic diagram of molecular docking of the components of supramolecular epigallocatechin gallate betaine cocrystal.

[0033] Figure 4 Schematic diagram of the interaction forces among the components of the supramolecular epigallocatechin gallate betaine cocrystal.

[0034] Figure 5 This is the XRD pattern of the supramolecular epigallocatechin gallate betaine cocrystal.

[0035] Figure 6 This is the stability observation diagram of supramolecular epigallocatechin gallate betaine and epigallocatechin gallate aqueous solution within 30 days.

[0036] Figure 7 SEM images of the dual supramolecular epigallocatechin gallate betaine raw material, components and physical mixture.

[0037] Figure 8 This is the stability observation diagram of the double supramolecular epigallocatechin gallate solid within 30 days.

[0038] Fig. 9 This is a stability observation diagram of dual supramolecular epigallocatechin gallate, supramolecular epigallocatechin gallate betaine and epigallocatechin gallate aqueous solution within 30 days.

[0039] Fig.10 Statistical graph of the permeation efficiency of dual supramolecular epigallocatechin gallate and epigallocatechin gallate.

[0040] Fig.11 This is a statistical graph of the PGE2 content secreted by human keratinocytes at a concentration of 0.05%.

[0041] Fig.12 This is a statistical chart of the relative expression of GM-CSF in mouse macrophages at a concentration of 0.08%.

[0042] Fig.13 These are microscopic images of fluorescent staining of human sebaceous gland cells at different concentrations.

[0043] Fig.14 This is a statistical graph of fluorescence quantification of human sebaceous gland cells under the action of different concentrations of epigallocatechin gallate.

[0044] Fig.15This is a statistical graph of fluorescence quantification of human sebaceous gland cells under the action of different concentrations of dual supramolecular epigallocatechin gallate.

[0045] Fig.16 This is a statistical graph of fluorescence quantification of human sebaceous gland cells under the action of different concentrations of supramolecular epigallocatechin gallate betaine.

[0046] Fig.17 This is the inhibition curve of tyrosinase (bisphenol) activity inhibition under the action of different concentrations of dual supramolecular epigallocatechin gallate.

[0047] Fig.18 This is the inhibition curve of tyrosinase (bisphenol) activity inhibition under the action of supramolecular epigallocatechin gallate betaine at different concentrations.

[0048] Fig.19 This is an inhibition curve of tyrosinase (bisphenol) activity inhibition under the action of different concentrations of epigallocatechin gallate.

[0049] Fig. 20 This is a statistical graph of the relative content of ROS under the action of different concentrations of dual supramolecular epigallocatechin gallate.

[0050] Fig.21 This is a statistical chart of the relative content of ROS under the action of different concentrations of supramolecular epigallocatechin gallate betaine.

[0051] Fig. 22 This is a statistical graph of the relative content of ROS in human skin fibroblasts under the action of different concentrations of epigallocatechin gallate.

[0052] Fig.23 This is a statistical graph of fluorescent staining of ROS content in human skin fibroblasts at different concentrations of samples. DETAILED DESCRIPTION

[0053] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those skilled in the art without making creative work are within the scope of protection of the present invention. Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as those commonly understood by those skilled in the art in the technical field of the present invention. The terms used in the description of the present invention in this specification are only for the purpose of describing specific implementation methods and are not intended to limit the present invention. The experimental methods in the following embodiments, unless otherwise specified, are all conventional methods. The experimental materials in the following embodiments, unless otherwise specified, are all purchased from conventional biochemical reagent stores:

[0054] Example 1. Supramolecular epigallocatechin gallate betaine supramolecular eutectic raw material

[0055] In order to solve the application problem of epigallocatechin gallate, this embodiment provides a supramolecular epigallocatechin gallate betaine supramolecular raw material, which is a pink solid powder. In this embodiment, epigallocatechin gallate is used as the target substrate, and potential ligands that can form cocrystals with it are preliminarily screened, such as organic bases including matrine, betaine, L-carnitine, etc.; organic acids including malic acid, tartaric acid, citric acid, etc. Betaine is selected as a potential ligand from the above two types of compounds to form a supramolecular cocrystal with epigallocatechin gallate.

[0056] In order to explore the hydrogen bonding sites of supramolecular epigallocatechin gallate betaine, molecular docking of epigallocatechin gallate and betaine was performed in this example, and the most stable conformation was selected as a reference among the generated conformations. The results are as follows: Figure 2 and Figure 3 As shown, Figure 2 This is the molecular distribution diagram of each component of the supramolecular epigallocatechin gallate betaine cocrystal. Figure 3 Schematic diagram of molecular docking of the components of supramolecular epigallocatechin gallate betaine cocrystal.

[0057] Figure 2 and Figure 3 The results show that according to its three-dimensional structure and computational simulation results, hydrogen bonds exist between epigallocatechin gallate and betaine. At the same time, the interaction force of supramolecular epigallocatechin gallate betaine was calculated and analyzed, and the results are as follows Figure 4 As shown, Figure 4 Schematic diagram of the interaction forces among the components of the supramolecular epigallocatechin gallate betaine cocrystal. Figure 4 The results showed that there were obvious hydrogen bonds, van der Waals interactions and other weak interactions between epigallocatechin gallate and betaine components, and various interactions jointly promoted the formation of supramolecular epigallocatechin gallate betaine.

[0058] The crystal structure of supramolecular epigallocatechin gallate betaine cocrystal, epigallocatechin gallate and betaine was analyzed by X-ray diffraction technique. Figure 5 shown. Figure 5The results showed that the supramolecular epigallocatechin gallate betaine had a 2θ angle of 5.7±0.2 compared with betaine and epigallocatechin gallate monomers. 0 , 8.9±0.2 0 , 10.8±0.2 0 , 21.9±0.2 0 and 26.3±0.2 0 The characteristic peaks appear at , indicating that there is obvious eutectic formation between the components. The results of 2θ angle, interplanar spacing d, and relative intensity (%) of supramolecular epigallocatechin gallate betaine X-ray diffraction are shown in Table 1.

[0059] Table 1 2θ angle, interplanar spacing d, relative intensity (%) of supramolecular epigallocatechin gallate betaine

[0060]

[0061] In order to study the stability of supramolecular epigallocatechin gallate betaine aqueous solution, the supramolecular epigallocatechin gallate betaine and epigallocatechin gallate monomer aqueous solution were treated at room temperature, high temperature (45℃, 50℃), low temperature (5℃), ultra-low temperature (-15℃) and sunlight for 30 days, and the changes in the appearance stability of the aqueous solution were observed. The appearance, color, odor and other indicators were used to test whether the supramolecular epigallocatechin gallate betaine and epigallocatechin gallate monomer aqueous solution were stable under various conditions.

[0062] The stability of supramolecular epigallocatechin gallate betaine and epigallocatechin gallate monomer aqueous solution was observed for 30 days. Figure 6 shown. Figure 6 The results showed that the supramolecular epigallocatechin gallate betaine aqueous solution had no obvious changes in appearance, color and odor after being treated at room temperature, high temperature (45℃, 50℃), low temperature (5℃), ultra-low temperature (-15℃) and sunlight for 15 days. After being placed for 30 days, it was treated with high temperature (45℃, 50℃) and sunlight, and its appearance changed slightly, and there was no obvious change in its appearance, color and odor at room temperature, low temperature (5℃) and ultra-low temperature (-15℃). The epigallocatechin gallate monomer aqueous solution changed its appearance color significantly after being treated with high temperature (45℃, 50℃) and sunlight for 30 days, and there was no obvious change in its appearance, color and odor at room temperature, low temperature (5℃) and ultra-low temperature (-15℃). By comparing the aqueous solutions of epigallocatechin gallate monomer and supramolecular epigallocatechin gallate betaine after 30 days of observation, it can be concluded that the supramolecular epigallocatechin gallate betaine is more stable than epigallocatechin gallate monomer.

[0063] Example 2: Preparation method of supramolecular epigallocatechin gallate betaine supramolecular raw material

[0064] This embodiment provides a method for preparing the supramolecular epigallocatechin gallate betaine supramolecular raw material of embodiment 1, combined with Figure 1 As shown, the preparation method of the supramolecular epigallocatechin gallate betaine supramolecular raw material of this embodiment specifically comprises the following steps:

[0065] S1: adding a solvent, epigallocatechin gallate and betaine into an ultrasonic reactor, starting ultrasound at a first reaction stirring speed and a first reaction temperature and continuing the reaction for a first reaction time to form a suspension of epigallocatechin gallate and betaine.

[0066] Specifically, the solvent can be selected from distilled water, anhydrous ethanol and ethyl acetate, among which anhydrous ethanol is preferred. Anhydrous ethanol has the advantages of moderate price, relatively efficient reaction and non-toxicity. The molar ratio of epigallocatechin gallate to betaine is 1:1. The ultrasonic power in the ultrasonic reactor is set to 1000~2000w, preferably 1800w; the ultrasonic frequency is set to 10~25KHz, preferably 20KHz; the first reaction stirring speed is set to 200~1000rpm, preferably 400~800rpm; the first reaction temperature is 30~70℃, preferably 30~50℃; the first reaction time is 1~8h, preferably 1~6h.

[0067] Ultrasonic reactor is a reactor that uses strong ultrasound to produce cavitation effect in liquid and ultrasonically treat substances. Ultrasonic wave is a sound wave with a frequency higher than 20,000 Hz. It has good directionality, strong penetration ability, and is easy to obtain concentrated sound energy. It uses mechanical effect to improve dispersion and enable heterogeneous reactions to proceed smoothly. This violent interaction between particles will cause the temperature of the liquid to rise suddenly, which plays a good stirring role, thereby accelerating chemical reactions; the secondary effects of ultrasound, such as mechanical vibration, emulsification, diffusion, and crushing, are conducive to the full mixing of reactants, which is more effective than the general one-way stirring effect.

[0068] S2: transferring the suspension to an evaporation crystallization reaction tank, cooling and crystallizing at a second reaction temperature to obtain supramolecular epigallocatechin gallate betaine.

[0069] Specifically, the second reaction temperature in the evaporation crystallization reaction tank is set to -10~20°C, preferably -10~5°C; at -10~5°C, the suspension is cooled and crystallized, and the cooling crystallization makes the solute precipitate from the solution in the form of crystals by lowering the temperature. By utilizing the principles of heat transfer and material transfer, the temperature and cooling rate are controlled to promote the solute in the solution to crystallize and deposit into solid particles.

[0070] The method for preparing supramolecular epigallocatechin gallate betaine of the invention has simple steps and can obtain supramolecular epigallocatechin gallate betaine without purification.

[0071] Example 3: Self-assembly of supramolecular raw materials from hydroxypropyl-β-cyclodextrin and supramolecular epigallocatechin gallate betaine

[0072] Taking supramolecular epigallocatechin gallate betaine as the target object, a host substance that can undergo host-guest self-assembly with it is preliminarily screened, such as macromolecular substances including cyclodextrin, chitosan, carboxymethyl cellulose, etc. A potential host substance is selected from the above macromolecular substances to undergo host-guest self-assembly with supramolecular epigallocatechin gallate betaine to form a supramolecular eutectic. In this embodiment, cyclodextrin and supramolecular epigallocatechin gallate betaine are selected to undergo host-guest self-assembly to form a supramolecular eutectic.

[0073] The hydroxypropyl-β-cyclodextrin and supramolecular epigallocatechin gallate betaine self-assembled supramolecular raw material of the present invention is composed of hydroxypropyl-β-cyclodextrin as the main body, and the supramolecular epigallocatechin gallate betaine as the guest substance for host-guest self-assembly under weak interactions such as hydrogen bonds and van der Waals interactions, that is, hydroxypropyl-β-cyclodextrin with a cavity structure is used to wrap the supramolecular epigallocatechin gallate betaine eutectic to form a supramolecular structure, so that epigallocatechin gallate and betaine form a eutectic with better stability and higher bioavailability while maintaining the original efficacy, and having the effects of anti-oxidation, whitening, soothing and oil control.

[0074] In one embodiment, the molar ratio of hydroxypropyl-β-cyclodextrin to the supramolecular epigallocatechin gallate betaine cocrystal is 1:1 to 1:5.

[0075] Example 4: Preparation method of self-assembled supramolecular raw material of hydroxypropyl-β-cyclodextrin and supramolecular epigallocatechin gallate betaine

[0076] This embodiment is used to provide a method for preparing the self-assembled supramolecular raw material of hydroxypropyl-β-cyclodextrin and supramolecular epigallocatechin gallate betaine of embodiment 3. On the basis of the preparation method of embodiment 2, Figure 1As shown, the specific steps include:

[0077] S3: adding a solvent, cyclodextrin and the supramolecular epigallocatechin gallate betaine of Example 1 into a reaction kettle, stirring for a second reaction time at a second reaction stirring speed and a third reaction temperature to form a suspension of hydroxypropyl-β-cyclodextrin and supramolecular epigallocatechin gallate betaine.

[0078] Specifically, the solvent can be selected from distilled water, anhydrous ethanol and ethyl acetate, among which distilled water is preferred. Distilled water has the advantages of being more economical and non-toxic. The molar ratio of hydroxypropyl-β-cyclodextrin to the supramolecular epigallocatechin gallate betaine of Example 1 is 1:1~1:5. The stirring speed of the second reaction is set to 200~1000rpm, preferably 600~1000rpm; the third reaction temperature is 20~50°C, preferably 25~45°C; the second reaction time is 1~12h, preferably 6~12h.

[0079] S4: transferring the suspension from the reactor to a high-pressure homogenizer, homogenizing the solution by high pressure, wherein the pressure of the homogenizing valve is 100-300 MPa, and performing dialysis after homogenization to form a dispersed stock solution of hydroxypropyl-β-cyclodextrin and supramolecular epigallocatechin gallate betaine;

[0080] Specifically, the homogenizing valve pressure is set to 100-300MPa, preferably 250MPa. The high-pressure homogenizer uses a high-pressure reciprocating pump as a power transmission and material conveying mechanism to convey the material to the working valve part. In the process of passing through the working valve, the material to be processed produces strong shearing, impact and cavitation under high pressure, so that the liquid substance or solid particles with liquid as a carrier are ultra-fine, and the suspension is homogenized to form a hydroxypropyl-β-cyclodextrin and supramolecular epigallocatechin gallate betaine dispersion stock solution.

[0081] S5: transferring the dispersed stock solution to a spray dryer, setting the feed temperature and spray speed for spray drying, and obtaining a self-assembled supramolecular raw material of hydroxypropyl-β-cyclodextrin and supramolecular epigallocatechin gallate betaine.

[0082] Specifically, spray drying is to disperse the material to be dried into very fine mist-like particles through mechanical action (increasing the water evaporation area and accelerating the drying process), and then contact with hot air to remove most of the water in an instant, so that the solid matter in the material is dried into powder. The feed temperature of spray drying is set to 100~200℃, preferably 150~180℃; the spraying speed is set to 5~15mL / min, preferably 8~10mL / min.

[0083] The feed temperature and spraying speed must be well controlled during spray drying. The inlet air temperature is the initial source of heat, so it is necessary to ensure that the atomized hydroxypropyl-β-cyclodextrin and supramolecular epigallocatechin gallate betaine self-assembled supramolecular dispersion stock solution can be in a dry or semi-dry state after air drying. At the same time, the inlet air temperature is also the biggest determining factor of the outlet air temperature. When other parameters remain unchanged, the outlet air temperature can be adjusted by controlling the inlet air temperature. The outlet air temperature determines the final state of the supramolecular raw material of hydroxypropyl-β-cyclodextrin and supramolecular epigallocatechin gallate betaine self-assembled. The spraying speed in spray drying directly determines the running trajectory and residence time of the material in the drying chamber, separator and conveyor, which can directly affect the product yield of the supramolecular raw material of hydroxypropyl-β-cyclodextrin and supramolecular epigallocatechin gallate betaine self-assembled.

[0084] Example 5: Morphological structure analysis of the supramolecular raw material of self-assembly of hydroxypropyl-β-cyclodextrin and supramolecular epigallocatechin gallate betaine

[0085] In order to explore the morphological structure of the supramolecular raw material self-assembled by hydroxypropyl-β-cyclodextrin and supramolecular epigallocatechin gallate betaine, the supramolecular raw material self-assembled by hydroxypropyl-β-cyclodextrin and supramolecular epigallocatechin gallate betaine was subjected to scanning electron microscopy. Figure 7 As shown, the physical mixture of epigallocatechin gallate, betaine, hydroxypropyl-β-cyclodextrin, hydroxypropyl-β-cyclodextrin and supramolecular epigallocatechin gallate betaine cocrystal and double supramolecular epigallocatechin gallate were analyzed by scanning electron microscopy, wherein, Figure 7 A to E in the figure represent scanning electron microscope images of hydroxypropyl-β-cyclodextrin, epigallocatechin gallate, betaine, physical mixture, and dual supramolecular epigallocatechin gallate, respectively.

[0086] The results showed that epigallocatechin gallate and betaine existed in the form of needle-like crystals, while hydroxypropyl-β-cyclodextrin existed in the form of amorphous spheres. In the micrograph of the physical mixture, the characteristic crystals of supramolecular epigallocatechin gallate betaine and the amorphous spheres of hydroxypropyl-β-cyclodextrin were found to exist simultaneously. The dual supramolecular epigallocatechin gallate appeared in the form of irregular particles, in which the original forms of both components disappeared, and amorphous small aggregates of irregular sizes existed. This result indicates that when supramolecular epigallocatechin gallate betaine and hydroxypropyl-β-cyclodextrin powders are simply mixed together, they do not form a tight bond and continue to exist in their original state, but when hydroxypropyl-β-cyclodextrin and supramolecular epigallocatechin gallate betaine host-guest self-assemble to form a dual supramolecular, they form a tight bond and epigallocatechin gallate and betaine no longer exist in a crystalline state.

[0087] Example 6: Study on the Stability of the Self-Assembled Supramolecular Raw Materials of Hydroxypropyl-β-Cyclodextrin and Epigallocatechin Gallate Betaine

[0088] In order to study the stability of the self-assembled supramolecular raw material of hydroxypropyl-β-cyclodextrin and supramolecular epigallocatechin gallate betaine, this example treated the double supramolecular epigallocatechin gallate solid at room temperature, high temperature (45°C), low temperature (5°C), ultra-low temperature (-15°C) and sunlight for 30 days, and then observed the change in the solid appearance stability. The appearance, color, odor and other indicators were used to test whether the double supramolecular epigallocatechin gallate solid powder was stable under various conditions. The results are as follows: Figure 8 shown.

[0089] The results showed that after 30 days of treatment at room temperature, high temperature (45℃), low temperature (5℃), ultra-low temperature (-15℃) and sunlight, there was no obvious change in appearance, color and odor, indicating that the dual supramolecular epigallocatechin gallate solid was stable under all conditions.

[0090] Example 7: Study on the stability of the aqueous solution of the supramolecular raw material self-assembled from hydroxypropyl-β-cyclodextrin and supramolecular epigallocatechin gallate betaine

[0091] While testing the stability of the double supramolecular epigallocatechin gallate solid, a comparative test was also conducted on the stability of its aqueous solution. In this embodiment, after treating the double supramolecular epigallocatechin gallate, supramolecular epigallocatechin gallate betaine, and epigallocatechin gallate monomer aqueous solutions at room temperature, high temperature (45°C, 50°C), low temperature (5°C), ultra-low temperature (-15°C), and sunlight for 30 days, the changes in the appearance stability of the aqueous solutions were observed. The appearance, morphology, color, odor and other indicators were used to test whether the double supramolecular epigallocatechin gallate, supramolecular epigallocatechin gallate betaine, and epigallocatechin gallate monomer aqueous solutions were stable under various conditions. The results are as follows: Fig. 9 shown.

[0092] The results showed that the stability of the dual supramolecular epigallocatechin gallate, supramolecular epigallocatechin gallate betaine, and epigallocatechin gallate monomer aqueous solution was observed for 30 days. Fig. 9 As shown, the appearance, color and odor of the double supramolecular epigallocatechin gallate did not change significantly after being treated at room temperature, high temperature (45℃, 50℃), low temperature (5℃), ultra-low temperature (-15℃) and sunlight for 30 days, indicating that the double supramolecular epigallocatechin gallate aqueous solution was stable under all conditions, and the appearance, color and odor did not change significantly; the supramolecular epigallocatechin gallate betaine aqueous solution was stable under room temperature, high temperature (45℃, 50℃), low temperature (5℃), ultra-low temperature (-15℃) After 15 days of treatment at 5℃ and sunlight, the appearance, color and smell did not change significantly. After 30 days of high temperature (45℃, 50℃) and sunlight treatment, the appearance changed slightly, and the appearance, color and smell did not change significantly at room temperature, low temperature (5℃) and ultra-low temperature (-15℃); after 30 days of high temperature (45℃, 50℃) and sunlight treatment, the appearance color of the aqueous solution of epigallocatechin gallate monomer changed significantly, and the appearance, color and smell did not change significantly at room temperature, low temperature (5℃) and ultra-low temperature (-15℃). Comparative observation of 30-day epigallocatechin gallate monomer, supramolecular epigallocatechin gallate betaine, and double supramolecular epigallocatechin gallate aqueous solution showed that the stability of their aqueous solutions was: double supramolecular epigallocatechin gallate > supramolecular epigallocatechin gallate betaine > epigallocatechin gallate monomer.

[0093] Example 8: Study on the transdermal penetration effect of the supramolecular raw material self-assembled from hydroxypropyl-β-cyclodextrin and supramolecular epigallocatechin gallate betaine

[0094] This example is used to study the transdermal penetration effect of the supramolecular raw material self-assembled from hydroxypropyl-β-cyclodextrin and supramolecular epigallocatechin gallate betaine. The raw material prepared in Example 2 was prepared into a double supramolecular epigallocatechin gallate solution with a concentration of 2.2%, and an epigallocatechin gallate solution of the corresponding concentration was prepared at the same time. The above solution was tested for transdermal effect, and the specific test method was as follows:

[0095] I. Microscopic examination: Select undamaged pig skin under a dissecting microscope, cut 6 pieces of skin of the same size, wash once with physiological saline, and absorb the surface moisture with filter paper.

[0096] II. Fix the skin: Fix the skin on the Franz diffusion cell with the stratum corneum facing the administration chamber and the dermis facing the receiving chamber. Add 17 mL of normal saline into the receiving chamber and remove bubbles to ensure that there are no bubbles between the dermis and the receiving solution.

[0097] III. Drug administration: Turn on the instrument in advance, adjust the water bath temperature to 32±1℃, add 1.0mL of drug into the drug administration chamber, seal with sealing film, and add tin foil to prevent liquid evaporation. The effective penetration area is 0.36πcm 2 .

[0098] IV. Infiltration: Set stirring speed to 300 r / min.

[0099] V. Sampling: Sampling was performed at specific time points (2h, 4h, 6h, 22h, 23h, 24h), filtered through a 0.22μm organic membrane, and then tested by HPLC. The cumulative permeation through the skin was calculated. The results are as follows: Fig.10 shown.

[0100] The results showed that after 24 hours of percutaneous penetration, the permeation per unit area of ​​2.2% dual supramolecular epigallocatechin gallate solution and the corresponding epigallocatechin gallate solution were 59.92 and 20.39 μg / cm, respectively, based on the epigallocatechin gallate content. 2 After 24 hours of percutaneous penetration, the permeation amount per unit area of ​​epigallocatechin gallate in the dual supramolecular epigallocatechin gallate solution was 2.94 times that of the corresponding epigallocatechin gallate solution. The results showed that the percutaneous penetration ability of the dual supramolecular epigallocatechin gallate was better than that of the epigallocatechin gallate monomer.

[0101] Comparative Example 1: The difference between this comparative example and Example 1 and Example 2 is that epigallocatechin gallate is used as the target substrate, and different ligands are screened to form a co-crystal therewith.

[0102] Under nitrogen protection, epigallocatechin gallate and matrine or tartaric acid were added to an ethanol solution at 45°C and 600rpm with continuous stirring. After sufficient ultrasonic stirring for 3 hours and evaporation and crystallization, it was found that the substance formed by epigallocatechin gallate and matrine or tartaric acid had an uneven appearance color and failed to successfully form the corresponding eutectic.

[0103] Comparative Example 2: The difference between the comparative example 2 and the examples 3 and 4 is that the supramolecular epigallocatechin gallate betaine is used as the target object, and different host substances are screened to form supramolecular eutectics capable of host-guest self-assembly therewith.

[0104] Under the protection of inert gas, chitosan and the supramolecular epigallocatechin gallate betaine cocrystal were added to distilled water, and the mixture was stirred for a certain period of time and then high-pressure homogenized. The mixture was then dialyzed for 3 times, and each dialysis was performed for 3 hours before spray drying. The solid stability of the formed solid substance was tested, and it was found that the host-guest self-assembly raw material formed by the chitosan and the supramolecular epigallocatechin gallate betaine cocrystal was unstable under high temperature conditions and discolored severely.

[0105] Test Example 1: Soothing Effects of Dual Supramolecular Epigallocatechin Gallate, Supramolecular Epigallocatechin Gallate Betaine, and Epigallocatechin Gallate Monomer

[0106] Evaluation of the soothing effect of the sample: By comparing the difference in the secretion of PGE2 by human keratinocytes after administration of the model control and the test substance, the effect of the test substance on inhibiting the secretion of PGE2 was evaluated. The PGE2 content was determined by enzyme-linked immunosorbent assay (ELISA) and the PGE2 content was calculated. The test scheme is shown in Table 2-1.

[0107] Testing basis: "Keratinocyte inflammatory factor (PGE2) release experimental method".

[0108] Result determination: The t-test method was used for analysis, with the test level α=0.05; P≥0.05 indicated no statistical difference, indicating that the sample had no effect on promoting changes in the content of inflammatory factors; P<0.05 indicated a significant difference and the content of inflammatory factors was significantly reduced, indicating that the sample had a soothing effect.

[0109] Specific experimental steps:

[0110] Ⅰ (Cell plating): Inoculate the human keratinocyte suspension into a 24-well plate and culture in an incubator for 24 hours.

[0111] Ⅱ (Preparation of solution): Prepare the working solution of the test substance according to the test plan (Table 2-1).

[0112] III (drug addition): According to the test plan (Table 2-1), drug addition was performed in groups, 1 mL was added to each well, 3 replicates were added to each group, and the effect was 2 hours. Except for the NC group, the culture medium was added, and the drug-containing culture medium containing LPS was added to the other groups.

[0113] IV (ELISA test): Collect the supernatant and perform the test according to the instructions of the ELISA kit.

[0114] The PGE2 content of the samples was determined on human keratinocytes, with dexamethasone as the control. The results of the relative content of PGE2 in different groups are shown in Table 2-2, and the comparison of the relative content of PGE2 is shown in Fig.11 .

[0115] Table 2-1 PGE2 test plan

[0116]

[0117] Table 2-2 Results of relative content of PGE2 in different samples at 0.05% concentration

[0118]

[0119] The results showed that compared with the NC group, the relative content of PGE2 in the M group was significantly increased (P<0.05), indicating that the stimulation conditions of this experiment were effective; compared with the M group, the relative content of PGE2 in the PC group was significantly decreased (P<0.05), indicating that this experiment was effective; compared with the M group, the relative content of PGE2 in the samples of dual supramolecular epigallocatechin gallate, supramolecular epigallocatechin gallate betaine, and epigallocatechin gallate were significantly decreased at a test concentration of 0.05% (P<0.05), and the inhibition rates were 80.10%, 69.54%, and 58.22%, respectively, indicating that dual supramolecular epigallocatechin gallate, supramolecular epigallocatechin gallate betaine, and epigallocatechin gallate can inhibit the secretion of PGE2 at a concentration of 0.05% and achieve a soothing effect.

[0120] In summary, at the sample concentration of 0.05%, the ability of supramolecular epigallocatechin gallate betaine to inhibit PGE2 release was better than that of epigallocatechin gallate, while the ability of dual supramolecular epigallocatechin gallate to inhibit PGE2 release was better than that of supramolecular epigallocatechin gallate betaine and epigallocatechin gallate (P<0.05), indicating that at this concentration, the soothing effect of dual supramolecular epigallocatechin gallate>supramolecular epigallocatechin gallate betaine>epigallocatechin gallate.

[0121] Comparative test example 1: Soothing effects of dual supramolecular epigallocatechin gallate, supramolecular epigallocatechin gallate betaine, and epigallocatechin gallate monomer

[0122] The difference between comparative test example 1 and test example 1 is that the soothing effect of the sample is evaluated by detecting the relative expression level of the GM-CSF gene.

[0123] Evaluation of the soothing effect of the sample: LPS induces macrophage activation cell model. After the sample acts on the cell model for a certain period of time, the relative expression level of GM-CSF gene is detected to evaluate whether the sample has a soothing effect.

[0124] Experimental system:

[0125] Cell system: mouse mononuclear macrophages (RAW264.7);

[0126] Cell generation number: at least 2 generations;

[0127] The number of cells per group: 3×10 5 Pieces / hole.

[0128] Specific experimental steps:

[0129] Ⅰ: Inoculate cells into 6-well plates (3×10 5 / well), incubated at 37°C, 5% CO2 for 24 h.

[0130] Ⅱ: Remove the culture medium and gently rinse the cells once or zero times with D-Hanks. Replace the culture medium with fresh medium for the normal control group, replace the culture medium with fresh medium containing modeling agent (LPS) and sample for the sample group, and replace the culture medium with fresh medium containing modeling agent (LPS) for the model control group. Continue to incubate at 37°C, 5% CO2 for 24h.

[0131] III: Extract total RNA from each experimental group, synthesize cDNA, and use q-PCR to detect β-actin and target gene expression. IV: Use β-actin as an internal reference for gene expression to calculate the relative RNA expression of the target gene.

[0132] IV: Use β-actin as the internal reference of gene expression to calculate the relative RNA expression of the target gene.

[0133] Relative RNA expression

[0134] 模型对照组 样品组

[0135] 目的基因

[0136] The samples were used to carry out the GM-CSF relative expression test on mouse macrophage RAW264.7. The results of the GM-CSF relative expression test in different groups are shown in Table 3-1. The results of the GM-CSF relative expression comparison are shown in Fig.12 shown.

[0137] Table 3-1 Detection results of relative expression of GM-CSF in different samples at 0.08% concentration

[0138]

[0139] The results showed that the GM-CSF gene expression levels of the samples of the dual supramolecular epigallocatechin gallate group, supramolecular epigallocatechin gallate betaine group, and epigallocatechin gallate group were significantly reduced compared with the model control group, revealing that the samples have a soothing effect. At a concentration of 0.08%, the relative expression levels of the GM-CSF gene in the samples of the dual supramolecular epigallocatechin gallate group and supramolecular epigallocatechin gallate betaine group were significantly different from those in the epigallocatechin gallate group.

[0140] In summary, at the sample concentration of 0.08%, the ability of supramolecular epigallocatechin gallate betaine to reduce the gene expression of GM-CSF was better than that of epigallocatechin gallate, and the ability of dual supramolecular epigallocatechin gallate to reduce the gene expression of GM-CSF was better than that of epigallocatechin gallate and supramolecular epigallocatechin gallate betaine (P<0.05), indicating that at this concentration, the soothing effect was: dual supramolecular epigallocatechin gallate > supramolecular epigallocatechin gallate betaine > epigallocatechin gallate.

[0141] Test Example 2: Oil Control Efficacy of the Prepared Dual Supramolecular Epigallocatechin Gallate, Supramolecular Epigallocatechin Gallate Betaine, and Epigallocatechin Gallate Monomer

[0142] Evaluation of the oil control and acne removal effect of the sample: In this experiment, the human sebaceous gland cell line SZ95 was selected for the construction of an in vitro acne model. The oil secretion of the cells simulated the sebum secretion of human skin. Nile red dye was used to perform fluorescent staining on neutral lipids. Fluorescent staining microscopy and fluorescent quantitative methods were used to qualitatively and quantitatively detect the oil content of sebaceous gland cells, thereby evaluating the oil control and acne removal effect of the sample to be tested. The test schemes for the fluorescent quantitative method and fluorescent staining microscopy are shown in Table 4-1 and Table 4-5, respectively.

[0143] Testing basis: SN / T2328-2009 “Keratinocyte test for acute toxicity of cosmetics”.

[0144] Result determination: The t-test method was used for analysis, and the test level α=0.05; P≥0.05 indicated that there was no statistical difference, indicating that the sample had no effect of inhibiting the change of neutral lipid content; P<0.05 indicated that there was a significant difference and the neutral lipid content was reduced, indicating that the sample had the effect of controlling oil and removing acne.

[0145] 1. Specific steps of neutral lipid fluorescence quantitative experiment:

[0146] Ⅰ: Inoculate the human sebaceous gland cell suspension into a 96-well cell culture plate and culture for 18-24 hours until the cells are 80% confluent.

[0147] Ⅱ: According to the test scheme (Table 4-1), add the samples of various concentrations and positive controls, incubate for 48 hours, discard the supernatant, wash twice with PBS, add Nile red dye to the experimental group, add FDA to the control group, incubate for 5 minutes, and detect the released fluorescence on a multifunctional microplate reader. 485nm, 494nm excitation wavelengths and 565nm, 523nm absorption wavelengths are used to detect the fluorescence intensity of Nile red and FDA, respectively.

[0148] Table 4-1 Neutral lipid fluorescence quantitative test scheme

[0149]

[0150]

[0151] The samples were tested for neutral lipid content in human sebaceous gland cells. The test results are shown in Table 4-2 to Table 4-4. The comparison results of the effects of the samples on the neutral lipid levels in human sebaceous gland cells are shown in Figure 13~Figure 15 shown.

[0152] Table 4-2 Effects of dual supramolecular epigallocatechin gallate on neutral lipid levels in human sebaceous gland cells

[0153]

[0154] Table 4-3 Effects of supramolecular epigallocatechin gallate betaine on neutral lipid levels in human sebaceous gland cells

[0155]

[0156] Table 4-4 Effect of epigallocatechin gallate on neutral lipid levels in human sebaceous gland cells

[0157]

[0158] The results showed that compared with the NC group, the relative content of neutral lipids in the M group was significantly increased (P<0.05), indicating that the stimulation conditions of this experiment were effective; compared with the M group, the relative content of neutral lipids in the PC group was significantly decreased (P<0.05), indicating that this experiment was effective; compared with the M group, the relative content of neutral lipids in the sample double supramolecular epigallocatechin gallate was significantly decreased at the test concentration of 0.024% and 0.006% (P<0.05), and the inhibition rates were 16.92% and 13.68%, respectively, indicating that the double supramolecular epigallocatechin gallate at the concentration of 0.024% and 0.006% can inhibit oil secretion and achieve the effect of oil control and acne removal; compared with the M group, the relative content of neutral lipids in the sample supramolecular epigallocatechin gallate betaine was significantly increased at the test concentration of 0.024%. The inhibition rates were 9.29%, indicating that supramolecular epigallocatechin gallate betaine at a concentration of 0.024% could inhibit oil secretion and achieve the effect of oil control and acne removal, while the relative content of neutral lipids did not change significantly at a test concentration of 0.006% for supramolecular epigallocatechin gallate betaine (P<0.05), indicating that supramolecular epigallocatechin gallate betaine had no inhibitory effect on the change of neutral lipid content at a concentration of 0.006%; compared with group M, the relative content of neutral lipids did not change significantly at the test concentrations of 0.024% and 0.006% for sample epigallocatechin gallate (P<0.05), indicating that epigallocatechin gallate had no inhibitory effect on the change of neutral lipid content at concentrations of 0.024% and 0.006%.

[0159] 2. Specific steps of neutral lipid fluorescence microscopy:

[0160] Ⅰ: Conventional cell culture: human sebaceous gland cells were inoculated in 35 mm culture dishes and cultured for 18-24 h until the cells were 80% confluent.

[0161] Ⅱ: According to the test scheme (Table 4-5), add test samples of various concentrations and positive controls, incubate for 48 hours, discard the supernatant, quickly wash twice with PBS, add 4% paraformaldehyde and fix at room temperature for 5 minutes, wash twice with PBS, add Nile red dye and incubate at 37℃ in dark for 15 minutes, wash twice with PBS, observe the cells under a fluorescence microscope, the excitation wavelength of the detection is 460~490nm, and the absorption wavelength is 565nm.

[0162] Table 4-5 Neutral lipid fluorescence microscopy test scheme

[0163]

[0164] The sample was tested for neutral lipid fluorescence microscopy on human sebaceous gland cells. The microscopic examination picture is as follows: Fig.16 shown.

[0165] The results showed that compared with the M group, the neutral lipid fluorescence intensity of the samples with a concentration of 0.024% and 0.006% of double supramolecular epigallocatechin gallate was significantly weakened, indicating that the double supramolecular epigallocatechin gallate can inhibit oil secretion at a concentration of 0.024% and 0.006%, achieving the effect of oil control and acne removal. Compared with the M group, the neutral lipid fluorescence intensity of the samples with a concentration of 0.024% of supramolecular epigallocatechin gallate betaine was significantly weakened, indicating that the double supramolecular epigallocatechin gallate can inhibit oil secretion at a concentration of 0.024%, achieving the effect of oil control and acne removal. Moreover, the degree of neutral lipid fluorescence attenuation of dual supramolecular epigallocatechin gallate is better than that of monomeric epigallocatechin gallate at the same concentration, indicating that at this concentration, the oil-control and acne-removing effects of dual supramolecular epigallocatechin gallate > supramolecular epigallocatechin gallate betaine > epigallocatechin gallate.

[0166] In summary, at the sample concentrations of 0.024% and 0.006%, the ability of the dual supramolecular epigallocatechin gallate to inhibit oil secretion was better than that of epigallocatechin gallate (P<0.05), indicating that at this concentration, the order of oil control and acne removal effect was: dual supramolecular epigallocatechin gallate > supramolecular epigallocatechin gallate betaine > epigallocatechin gallate.

[0167] Test Example 3: Whitening Effects of the Prepared Dual Supramolecular Epigallocatechin Gallate and Epigallocatechin Gallate Monomer

[0168] Evaluate the whitening effect of samples: In the biosynthesis of melanin in the skin, tyrosinase is a key enzyme that acts on DOPA to form DOPA quinone, which spontaneously undergoes a series of reactions to finally form melanin. In pH 6.8 NaHPO4-citrate buffer, tyrosinase can catalyze the conversion of DOPA into DOPA quinone, and the absorbance value can be measured at 475nm on a spectrophotometer. Raw materials with tyrosinase activity inhibition can reduce the conversion of DOPA into DOPA quinone, thereby reducing the absorbance value. Based on the change in absorbance value, the inhibitory effect of the raw material on tyrosinase activity is evaluated.

[0169] Test basis: T / SHRH015-2018 "Cosmetics-Tyrosinase Activity Inhibition Test Method".

[0170] Result determination: The t-test method was used for analysis, with the test level α=0.05; P≥0.05 means no statistical difference, indicating no inhibitory effect on tyrosinase; P<0.05 means significant difference, indicating an inhibitory effect on tyrosinase, i.e., whitening effect. The smaller the sample IC50, the stronger the inhibitory effect on tyrosinase.

[0171] Specific steps of the experiment:

[0172] Set up sample tubes (T), sample background (T0), enzyme reaction tubes (C) and solvent background (C0). For each sample tube (T) of each tested concentration, three parallel tubes need to be set up. Add 1 mL of sample solution of the same concentration to each sample tube (T) and sample background (T0). Add 0.5 mL of tyrosinase solution to each sample tube (T) and enzyme reaction tube (C), mix well, and incubate in a 37°C water bath for 10 minutes. Add 2 mL of L-DOPA solution to each tube in turn, react for 5 minutes, and immediately transfer the reaction solution of each tube into a cuvette to measure the absorbance at 475 nm.

[0173] Tyrosinase inhibition rate (%)

[0174] The samples were tested for tyrosinase (bisphenol) activity, and the test results are shown in Table 5-1 to Table 5-3.

[0175] Table 5-1 Inhibition rate of dual supramolecular epigallocatechin gallate on tyrosinase (bisphenol) activity

[0176]

[0177] Table 5-2 Inhibition rate of supramolecular epigallocatechin gallate betaine on tyrosinase (bisphenol) activity

[0178]

[0179] Table 5-3 Inhibition rate of epigallocatechin gallate on tyrosinase (bisphenol) activity

[0180]

[0181] The results of the sample inhibition rate on tyrosinase (bisphenol) activity are shown in Table 5-1 to Table 5-3. The inhibition curve is as follows: Figure 18~Figure 20 shown.

[0182] The results showed that the sample dual supramolecular epigallocatechin gallate had an inhibition rate concentration of tyrosinase (bisphenol) activity IC50 = 4.93% (95% confidence interval IC50 = 4.69% ~ 5.18%), which has a whitening effect; the sample supramolecular epigallocatechin gallate betaine had an inhibition rate concentration of tyrosinase (bisphenol) activity IC50 = 6.63% (95% confidence interval IC50 = 6.10% ~ 7.18%), which has a whitening effect; the sample epigallocatechin gallate had an inhibition rate concentration of tyrosinase (bisphenol) activity IC50 = 9.15% (95% confidence interval IC50 = 8.49% ~ 9.82%), which has a whitening effect. Moreover, the dual supramolecular epigallocatechin gallate and supramolecular epigallocatechin gallate betaine inhibited the activity of bisphenol tyrosinase more strongly than the monomer epigallocatechin gallate at the same concentration. Therefore, it can be seen that the whitening effect is: dual supramolecular epigallocatechin gallate > supramolecular epigallocatechin gallate betaine > epigallocatechin gallate.

[0183] Test Example 4: Antioxidant efficacy of the prepared dual supramolecular epigallocatechin gallate, supramolecular epigallocatechin gallate betaine, and epigallocatechin gallate monomer

[0184] Evaluation of the antioxidant effect of samples: Reactive oxygen is considered to be an important cause of skin aging, which can cause thinning of the skin and wrinkles. Ultraviolet radiation can lead to the formation of intracellular reactive oxygen. The antioxidant effect of the sample can be reflected by detecting the ROS content in human skin fibroblasts. See Table 6-1 for the specific test plan.

[0185] Testing basis: T / GDCA014-2022 "Cellular Evaluation Method for the Evaluation of Antioxidant Activity of Cosmetics".

[0186] Result determination: The t-test method was used for analysis, with the test level α=0.05; P≥0.05 means there is no statistical difference, indicating that there is no significant change in the active oxygen content; P<0.05, the active oxygen content decreases and there is a significant difference, that is, the test substance has antioxidant effect.

[0187] Specific steps of the experiment:

[0188] Ⅰ: Inoculate the human skin fibroblast suspension into a 24-well plate and incubate for 24 hours.

[0189] II: Solution preparation: Dispense the working solution of the test substance according to the test plan (Table 6-1).

[0190] III: Drug administration: According to the test scheme in Table 6-1, the drugs were added in groups, and UVA irradiation was performed 4 hours later, with an irradiation dose of 4.8 J / cm 2After UVA irradiation, each well was replaced with fresh medium containing the test substance and cultured for 20 h.

[0191] IV: Detection: Wash the cells in each well twice with serum-free DMEM culture medium, add 10ųmol / L ROS dye DCFH-DA, incubate at 37℃ in the dark for 20min, wash again with serum-free culture medium for 3 times, and take pictures of one field of view randomly selected from each well under a fluorescence microscope with a 20x objective lens, with 3 replicates in each group.

[0192] Table 6-1 Antioxidant efficacy test plan

[0193]

[0194] The ROS content of the samples was tested on human skin fibroblasts. The test results are shown in Table 6-2~Table 6-3; the relative content of ROS on human skin fibroblasts between the groups is shown in Figure 20~Figure 22 As shown, the fluorescence microscopy of ROS content in human skin fibroblasts of the sample is as follows Fig.23 shown.

[0195] Table 6-2 Test results of ROS content of dual supramolecular epigallocatechin gallate

[0196]

[0197] Table 6-3 Test results of ROS content of supramolecular epigallocatechin gallate betaine

[0198]

[0199] Table 6-4 Detection results of ROS content in epigallocatechin gallate

[0200]

[0201] The results showed that compared with the NC group, the relative content of ROS in the M group increased significantly (P<0.05), indicating that the stimulation conditions of this experiment were effective; compared with the M group, the relative content of ROS in the PC group decreased significantly (P<0.05), indicating that this experiment was effective; compared with the M group, the relative content of ROS in the sample double supramolecular epigallocatechin gallate at 0.02% and 0.01% test concentrations decreased significantly (P<0.05), and the inhibition rates were 70.54% and 68.02%, respectively, indicating that the double supramolecular epigallocatechin gallate at 0.02% and 0.01% concentrations can inhibit ROS secretion and achieve antioxidant effect; compared with the M group, the sample supramolecular epigallocatechin gallate At the test concentrations of 0.02% and 0.01% of epigallocatechin gallate betaine, the relative content of ROS decreased significantly (P<0.05), and the inhibition rates were 65.27% and 60.76%, respectively, indicating that supramolecular epigallocatechin gallate betaine can inhibit ROS secretion at the concentrations of 0.02% and 0.01%, and achieve antioxidant effect; compared with group M, at the test concentrations of 0.02% and 0.01% of epigallocatechin gallate, the relative content of ROS decreased significantly (P<0.05), and the inhibition rates were 54.34% and 50.32%, respectively, indicating that epigallocatechin gallate can inhibit ROS secretion at the concentrations of 0.02% and 0.01%, and achieve antioxidant effect.

[0202] In summary, at the concentrations of 0.02% and 0.01%, the ability of the dual supramolecular epigallocatechin gallate to inhibit ROS secretion was better than that of supramolecular epigallocatechin gallate betaine and epigallocatechin gallate (P<0.05), and the ability of supramolecular epigallocatechin gallate betaine to inhibit ROS secretion was better than that of epigallocatechin gallate (P<0.05), indicating that at this concentration, the antioxidant effect was: dual supramolecular epigallocatechin gallate > supramolecular epigallocatechin gallate betaine > epigallocatechin gallate.

[0203] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technical personnel in this field can make some changes or modify the technical contents disclosed above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any brief modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A method for preparing a supramolecular raw material for self-assembly of hydroxypropyl-β-cyclodextrin and supramolecular epigallocatechin gallate betaine, characterized in that: The steps include: S1. Add a solvent and epigallocatechin gallate and betaine in a molar ratio of 1:1-1:3 into an ultrasonic reactor, apply ultrasonic energy at a first reaction stirring speed of 400-800 rpm and a first reaction temperature of 30-50° C., an ultrasonic power of 1000-2000 w, an ultrasonic frequency of 10-25 kHz, and continue the reaction for 1-6 hours to form a suspension of epigallocatechin gallate and betaine; S2, transferring the suspension to an evaporation crystallization reaction tank, and cooling and crystallizing at a second reaction temperature of -10 to 5°C to obtain supramolecular epigallocatechin gallate betaine eutectic; S3, adding a solvent and a hydroxypropyl-β-cyclodextrin and supramolecular epigallocatechin gallate betaine cocrystal in a molar ratio of 1:1 to 1:5 into a reactor, stirring the second reaction at a second reaction speed of 600 to 1000 rpm and a third reaction temperature of 25 to 45° C. for a second reaction time of 6 to 12 hours to form a suspension of hydroxypropyl-β-cyclodextrin and supramolecular epigallocatechin gallate betaine; S4, transferring the suspension from the reactor to a high-pressure homogenizer, homogenizing the solution by high pressure, wherein the pressure of the homogenizing valve is 100-300 MPa, and performing dialysis treatment after homogenization to form a dispersed stock solution of hydroxypropyl-β-cyclodextrin and supramolecular epigallocatechin gallate betaine; S5. Transfer the dispersed stock solution to a spray dryer, set the feed temperature and spray speed for spray drying, and obtain a self-assembled supramolecular raw material of hydroxypropyl-β-cyclodextrin and supramolecular epigallocatechin gallate betaine.

2. The method for preparing the self-assembled supramolecular raw material according to claim 1, characterized in that: The solvent in step S1 is anhydrous ethanol; the solvent in step S3 is distilled water.

3. The method for preparing the self-assembled supramolecular raw material according to claim 1, characterized in that: In step S4, the dialysis treatment uses a cellulose dialysis bag, and the molecular weight of the cellulose dialysis bag is 500-1000; the dialysis treatment time is 2-4 hours, and the number of dialysis is 1-3 times.

4. The method for preparing the self-assembled supramolecular raw material according to claim 1, characterized in that: The feed temperature of the spray drying process in step S5 is set to 150-180° C., and the spraying speed of the spray drying process is 8-10 mL / min.

5. A self-assembled supramolecular raw material of hydroxypropyl-β-cyclodextrin and supramolecular epigallocatechin gallate betaine prepared by the preparation method according to any one of claims 1 to 4.

6. The self-assembled supramolecular raw material of hydroxypropyl-β-cyclodextrin and supramolecular epigallocatechin gallate betaine according to claim 5, characterized in that: The supramolecular epigallocatechin gallate betaine cocrystal in the self-assembled supramolecular raw material is encapsulated in the cavity of hydroxypropyl-β-cyclodextrin; the molar ratio of hydroxypropyl-β-cyclodextrin to the supramolecular epigallocatechin gallate and betaine cocrystal is 1:1-1:

5.

7. The self-assembled supramolecular raw material of hydroxypropyl-β-cyclodextrin and supramolecular epigallocatechin gallate betaine according to claim 6, characterized in that: The supramolecular epigallocatechin gallate betaine eutectic is a supramolecular eutectic formed by epigallocatechin gallate and betaine through hydrogen bonding and van der Waals interactions. The structural formula of the supramolecular epigallocatechin gallate betaine is: ； The molar ratio of epigallocatechin gallate to betaine in the supramolecular eutectic is 1:1-1:

3.

8. The self-assembled supramolecular raw material of hydroxypropyl-β-cyclodextrin and supramolecular epigallocatechin gallate betaine according to claim 7, characterized in that: In the X-ray diffraction pattern of the supramolecular epigallocatechin gallate betaine cocrystal, at a 2θ angle of 5.7±0.2 0 , 8.9±0.2 0 , 10.8±0.2 0 , 21.9±0.2 0 and 26.3±0.2 0 , or at the crystal plane spacing d of 15.63±0.2Å, 9.91±0.2Å, 8.18±0.2Å, 4.06±0.2Å, 3.57±0.2Å, and 3.39±0.2Å, characteristic peaks with relative intensities of 4.9%, 8.2%, 4.7%, 44.4%, 100.0%, and 83.0% appear, respectively.

9. Use of the self-assembled supramolecular raw material of hydroxypropyl-β-cyclodextrin and supramolecular epigallocatechin gallate betaine as claimed in any one of claims 5 to 8 in the preparation of products in the fields of medicine and daily chemicals.

Images