Toxifolin compound, preparation method thereof and application of taxifolin compound in fields of relieving, oil control and aging resistance

Abstract

The invention belongs to the technical field of taxifolin, and discloses a taxifolin compound, a preparation method thereof and application of the taxifolin compound in the fields of relieving, oil control and senility .The taxifolin compound contains modified beta-cyclodextrin, taxifolin and auxiliary components, and the modified beta-cyclodextrin is modified through a polar group; the auxiliary component is selected from one or more of a hydrophilic polymer, an antioxidant and a targeting carrier. The taxifolin compound provided by the invention is high in inclusion rate and good in water solubility, the polar group modified beta-cyclodextrin can finely adjust the space structure of a cavity to expand an opening, the polar group modified beta-cyclodextrin has high matching degree with the molecular structure of taxifolin, a hydrophobic aromatic ring of taxifolin can enter the cavity to be combined with a hydrophobic core, and the taxifolin compound is high in inclusion rate and good in water solubility. The water solubility is good. The taxifolin compound provided by the invention is strong in anti-inflammatory ability, has a remarkable soothing effect, and also has excellent anti-aging and oil-control effects.

Description

Technical Field

[0001] This invention relates to the field of taxonasin complex technology, and more specifically, to a taxonasin complex and its preparation method, and its application in the fields of soothing, oil control, and anti-aging. Background Technology

[0002] Taxibberellin is a dihydroflavonoid compound extracted from pine plants. It has strong antioxidant properties, can scavenge free radicals, and also has anti-inflammatory and antiviral biological activities. It is widely used in the fields of medicine, health products, and cosmetics. Specifically in the field of cosmetics, taxibberellin can soothe and repair, and delay skin aging.

[0003] Taxobrin possesses unique chemical structural characteristics, resulting in low purity and poor stability. Its molecular structure is easily damaged by light, and the active ingredient is readily decomposed in strongly acidic or alkaline environments. Current techniques often use β-cyclodextrin to encapsulate taxobrin to prepare high-purity taxobrin complexes, which can improve the stability of taxobrin to some extent; however, the following drawbacks still exist: (1) Low inclusion rate of the taxonomic compound. Taxonomic compound molecules contain a hydrophobic aromatic ring and multiple phenolic hydroxyl groups. The hydrophobic aromatic ring needs to bind to the hydrophobic core of the β-cyclodextrin cavity, and the multiple phenolic hydroxyl groups need to form hydrogen bonds with the hydroxyl groups on the inside of the β-cyclodextrin cavity. However, the β-cyclodextrin cavity is small in size and easily oxidized and deformed, resulting in limited molecular structure matching with taxonomic compound, leading to a low inclusion rate of the taxonomic compound.

[0004] (2) The taurine complex has poor water solubility. The outer side of the β-cyclodextrin molecule is mainly composed of hydroxyl groups (-OH). These hydroxyl groups easily aggregate through hydrogen bonds, forming larger particles. Therefore, it has poor dispersibility in water and weak hydrophilicity, with a solubility of only 18.5 g / L at room temperature, indicating poor water solubility. The poor water solubility of β-cyclodextrin leads to the poor water solubility of the taurine complex formed by its encapsulation of taurine molecules.

[0005] This invention provides a piperidine complex with high inclusion rate or good water solubility, which is superior to the prior art.

[0006] Another object of the present invention is to provide a method for preparing the said piperidine complex.

[0007] Another object of the present invention is to provide the use of the said taurine complex in the preparation of cosmetics.

[0008] To solve the above-mentioned technical problems, the technical solution provided by the present invention is as follows: A taxine complex comprising modified β-cyclodextrin, taxine, and an auxiliary component, wherein the modified β-cyclodextrin is modified with a polar group; and the auxiliary component is selected from one or more of hydrophilic polymers, antioxidants, and targeting carriers.

[0009] The piperidine complex of the present invention is encapsulated with modified β-cyclodextrin, and the addition of auxiliary components has the following technical effects: (1) The inclusion rate of the taxone complex is high. On the one hand, the polar group modification of β-cyclodextrin can increase the basic sites on the inner side of its cavity, which can easily form hydrogen bonds with the phenolic hydroxyl groups of taxone. Moreover, the polar group modification can fine-tune the spatial structure of the cavity to expand the opening, which has a high degree of matching with the molecular structure of taxone, and facilitates the entry of the hydrophobic aromatic ring of taxone into the cavity to bind with the hydrophobic core, resulting in a high inclusion rate of the taxone complex. On the other hand, the hydrophilic polymer (such as PVP) in the auxiliary components can adjust the conformation of cyclodextrin, making the cavity opening more open, which has a high degree of matching with the molecular structure of taxone. In addition, the antioxidant maintains the structure of taxone and avoids oxidation that causes the structure of taxone to deform, ensuring the molecular structure matching between taxone and β-cyclodextrin. The targeting carrier increases the contact frequency between taxone and cyclodextrin through the "anchoring" effect, so the inclusion rate of the taxone complex is high.

[0010] (2) The taxonomic compound has good water solubility. On the one hand, the modified β-cyclodextrin introduces a highly polar group, which enhances the hydrophilicity of β-cyclodextrin. Moreover, the polar group of β-cyclodextrin can prevent the aggregation of hydroxyl groups through hydrogen bonds due to charge repulsion. On the other hand, the hydrophilic polymer in the auxiliary component enhances the water solubility of the taxonomic compound by forming a hydration layer. Furthermore, the steric hindrance of the hydrophilic polymer further prevents the aggregation of β-cyclodextrin, resulting in good water solubility of the taxonomic compound.

[0011] Preferably, the polar group is selected from amino groups, sulfonyl groups, carboxyl groups, quaternary ammonium salt groups, glycosyl groups, or polyethylene glycol chains.

[0012] The carboxyl group mentioned in this invention is specifically carboxymethyl.

[0013] More preferably, the polar group is selected from carboxyl, amino, or sulfonyl groups.

[0014] Preferably, the hydrophilic polymer is selected from polyvinylpyrrolidone, hydroxypropyl methylcellulose, chitosan, dextran, methoxy polyethylene glycol, or polyethylene glycol-polylysine copolymer.

[0015] Preferably, the antioxidant is selected from vitamin E, glutathione, tea polyphenols, coenzyme Q10, or lipoic acid.

[0016] Preferably, the targeting vector is selected from targeting peptides, folic acid, monoclonal antibodies, monoclonal antibody fragments, aptamers, or nanocarriers.

[0017] Preferably, the mass ratio of the piperidine, modified β-cyclodextrin and auxiliary components is 1:(4~8):(5~9).

[0018] Preferably, the auxiliary components in the piperidine complex of the present invention are a hydrophilic polymer, an antioxidant and a targeting carrier, in a mass ratio of (2~3):(1~2):(1~3).

[0019] When multiple auxiliary ingredients are used in combination, they can produce a "1+1>2" effect: Taxiardin + hydrophilic polymer + antioxidant. The hydration film of the hydrophilic polymer increases the moisture of the skin surface and promotes the penetration of taxiardin into the stratum corneum. Moreover, the hydrophilic polymer binds to the mucopolysaccharides in the dermis, which prolongs the retention time of taxiardin in the dermis. Taxiardin protects fibroblasts, and antioxidants repair the epidermal barrier, synergistically enhancing anti-aging and skin barrier repair.

[0020] This invention also provides a method for preparing the piperidine complex according to any one of the above claims, comprising the following steps: S1. Inclusion of Taxodiacetic acid: Modified β-cyclodextrin and taxodiacetic acid are added to a solvent in a certain proportion and stirred at 30~50℃ for 2~4 hours to form an inclusion suspension; S2. Mixing auxiliary components: Add auxiliary components to the inclusion complex suspension, stir evenly, and dry to obtain the taurine complex.

[0021] The present invention also provides the use of the taxanein complex described in any of the above claims in the preparation of cosmetics.

[0022] Preferably, the cosmetic has antioxidant, soothing, anti-aging and oil-controlling effects. Attached Figure Description

[0023] Figure 1 The effect of the test substance on MMP-1 (mean, n=3); Figure 2 The effect of the test substance on the expression of IL-6 mRNA (mean, n=3); Figure 3 The effect of the test substance on the expression of IL-1β mRNA (mean, n=3); Figure 4 The effect of the test substance on the related expression of TNF-α mRNA (mean, n=3); Figure 5 The effect of the test substance on lipid accumulation (mean, n=3). Detailed Implementation

[0024] The present invention will be further described in detail below with reference to specific embodiments.

[0025] Example 1 A taxane complex comprising modified β-cyclodextrin, taxane, and auxiliary components, wherein the modified β-cyclodextrin is modified with a sulfonyl (-SO3H) polar group; the auxiliary components are glucose, vitamin E, and folic acid; the mass ratio of taxane, modified β-cyclodextrin, and auxiliary components is 1:6:8, and the mass ratio of dextran, vitamin E, and folic acid is 2:1.5:2.

[0026] Among them: the hydrophilic polymer dextran (neutral hydrophilic polysaccharide) can prevent the aggregation of the complex through steric hindrance, thus enhancing water solubility; the hydrophobic side chain (isoprene chain) of vitamin E can be embedded in the cyclodextrin cavity to form a "bimolecular inclusion" with taurine, enhancing the steric stability of the inclusion complex. Its phenolic hydroxyl group can form a weak electrostatic attraction with -SO3⁻, which helps stabilize the inclusion structure. At the same time, its antioxidant effect protects the structural stability of taurine and improves the inclusion rate; folic acid contains carboxyl (-COOH) and amino (-NH2). After the carboxyl group dissociates, it carries a negative charge and can form a "charge dispersion effect" with -SO3⁻, reducing the local negative charge density, thus enhancing water solubility. The hydrophobic part of its pteridine ring can synergistically enter the cyclodextrin cavity with taurine, enhancing the inclusion effect.

[0027] The preparation method of the sulfonyl (-SO3H) modified β-cyclodextrin includes the following steps: (1) Sulfonation reaction: Add 80 mL of pyridine to 10 g of β-cyclodextrin and stir until completely dissolved to obtain solution A; then place solution A in an ice-water bath (0-5℃) and slowly add 5-8 mL of chlorosulfonic acid under magnetic stirring. The molar ratio of β-cyclodextrin to chlorosulfonic acid is about 1:6-8. The addition time is controlled within 30 minutes. After the addition is completed, keep the reaction in an ice-water bath and stir for 3 hours to allow the hydroxyl groups to fully combine with the sulfonyl groups; after the reaction is completed, slowly add deionized water to terminate the reaction, and then add 10% sodium hydroxide solution to adjust the pH to 7-8 to obtain the reaction solution; (2) Precipitation and purification: The reaction solution was slowly poured into anhydrous ethanol and stirred to produce a white precipitate. After standing for 1 hour, the precipitate was filtered, collected, and washed with anhydrous ethanol to obtain the crude product. (3) Dissolve the crude product in deionized water, concentrate it to 1 / 3 of the original volume by rotary evaporation, and dry it under vacuum at 60°C for 8 hours to obtain sulfonyl-modified β-cyclodextrin.

[0028] The preparation method of the taxanein complex includes the following steps: S1. Inclusion of Taxodiacetic glycosides: Sulfonyl-modified β-cyclodextrin was dissolved in PBS solution and stirred until completely dissolved; then, the ethanol solution of taxodiacetic glycosides was slowly added dropwise to the cyclodextrin solution in proportion, and the mixture was magnetically stirred at 40°C for 3 hours (300 r / min), followed by ultrasonic treatment at 300 W for 20 minutes to promote host-guest inclusion and form a suspension of taxodiacetic glycosides-cyclodextrin inclusion complex.

[0029] S2. Mixing auxiliary components: Add auxiliary components to the inclusion complex solution, stir evenly, and dry to obtain the tamarindin complex, specifically: S21 auxiliary component dispersion: Dissolve dextran in PBS and stir until completely dissolved to obtain a dextran solution; Add vitamin E to Tween 80 and PBS, and sonicate at 200W for 10 minutes to obtain a vitamin E emulsion. Dissolve folic acid in PBS, adjust the pH to 6.5-7.0, and stir until clear to obtain a folic acid solution; S22. Complex assembly: The above dextran solution, vitamin E emulsion, and folic acid solution were added sequentially to the taurine-cyclodextrin inclusion complex suspension, and the mixture was magnetically stirred at 30°C for 1 hour to ensure that the auxiliary components were evenly dispersed and combined with the inclusion complex to obtain a mixture. S23. Freeze-drying: Pour the mixture into a freeze-drying bottle, pre-freeze at -80℃ for 2 hours, and then freeze-dry at a vacuum degree ≤10Pa and a temperature of -50℃ for 48 hours to obtain the piperidine complex.

[0030] Example 2 This embodiment is the second embodiment of the present invention. Unlike embodiment 1, the polar group in the taurine complex is carboxymethyl, and the auxiliary components are chitosan, tea polyphenols and nanocarriers. The mass ratio of taurine, modified β-cyclodextrin and auxiliary components is 1:6:7, and the mass ratio of chitosan, tea polyphenols and nanocarriers is 2:2:1.

[0031] Among them: chitosan contains a large number of free amino groups (-NH2), which can form electrostatic attraction with the -COO⁻ of the carboxyl group, reducing the aggregation between cyclodextrin molecules and improving water solubility. In addition, its hydroxyl groups can also form hydrogen bonds with the phenolic hydroxyl groups of taurine, which helps to stabilize the inclusion complex. Tea polyphenols contain multiple phenolic hydroxyl groups, which can form hydrogen bonds with the carboxyl group (-COOH). In addition, its hydrophobic group (benzene ring) can be partially embedded in the cyclodextrin cavity, forming a "synergistic inclusion" with taurine, which enhances the stability of the inclusion complex. Moreover, the antioxidant properties of tea polyphenols can protect taurine from oxidation. The phospholipid bilayer of nanoliposomes can encapsulate the carboxyl-modified cyclodextrin-taurine inclusion complex. The hydrophilic groups on the surface of the liposomes (such as the polar head of phosphatidylcholine) can enhance water solubility. In addition, the encapsulation effect of the liposomes reduces the dissociation of the inclusion complex in water and improves the inclusion rate.

[0032] The preparation method of the carboxymethyl modified β-cyclodextrin includes the following steps: (1) Hydroxyl activation: Add deionized water to β-cyclodextrin and stir until completely dissolved; then slowly add NaOH solution and stir at 30°C for 30 minutes to deprotonate and activate the hydroxyl groups of β-cyclodextrin to generate -O⁻Na⁺; (2) Carboxymethylation reaction: Add chloroacetic acid to the (1) system, the molar ratio of β-cyclodextrin to chloroacetic acid is about 1:8-10, heat to 55℃, and stir magnetically for 2.5 hours; (3) Neutralization and precipitation: After the reaction is complete, cool to room temperature, adjust the pH of the solution to 7.0, and then slowly pour the solution into isopropanol. Stir to produce a white flocculent precipitate. After standing for 1 hour, filter and collect the precipitate. (4) Purification and drying: The precipitate was redissolved in deionized water, activated carbon was added and stirred at 60°C for 30 minutes to decolorize, and then filtered while hot; the filtrate was poured into isopropanol again to precipitate, and the precipitate was washed with isopropanol after filtration; finally, the precipitate was placed in a vacuum drying oven at 60°C for 10 hours to obtain carboxymethyl modified β-cyclodextrin.

[0033] The preparation method of the taxanein complex includes the following steps: S1. Inclusion of Taxonitrile: Carboxymethyl modified β-cyclodextrin was dissolved in PBS solution and stirred until completely dissolved; then, the ethanol solution of taxonitrile was slowly added dropwise to the cyclodextrin solution in proportion, and the mixture was magnetically stirred at 30°C for 4 hours (300 r / min), and ultrasonically treated with a power of 250 W for 15 minutes to promote host-guest inclusion and form a taxonitrile-cyclodextrin inclusion complex suspension; S2. Mixing auxiliary components: Add auxiliary components to the inclusion complex solution, stir evenly, and dry to obtain the tamarindin complex, specifically: S21 auxiliary component dispersion: Dissolve chitosan in dilute hydrochloric acid solution and stir until completely dissolved to obtain chitosan solution; Add tea polyphenols to deionized water, operate in the dark to prevent oxidation, and sonicate for 10 minutes to obtain a tea polyphenol solution. Soybean lecithin and cholesterol (mass ratio 3:1) were dissolved in chloroform, and the chloroform was removed by rotary evaporation (40℃, vacuum degree 0.08MPa) to form a uniform lipid film. mL of PBS (pH 6.0) was added, and the film was hydrated by shaking in a water bath at 37℃ for 30 minutes. After ultrasonic treatment (power 400W, time 10 minutes), it was processed by a high-pressure homogenizer (800 bar, 3 times) to obtain a nanoliposome suspension with a particle size of 100-200nm. S22. Complex assembly: Chitosan solution was slowly added dropwise to the piperidine-cyclodextrin inclusion complex solution at a rate of 1 mL / min, and the mixture was magnetically stirred at 30°C for 30 minutes; then tea polyphenol solution was added under light-protected conditions, and stirring was continued for 20 minutes. Finally, the nanoliposome suspension was added and incubated at 37°C for 40 minutes to obtain the complex suspension.

[0034] S23. Freeze-drying: Filter the suspension through a 0.22μm filter membrane, pour it into a freeze-drying bottle, pre-freeze at -40℃ for 3 hours, and freeze-dry (vacuum degree ≤10Pa, temperature -50℃, 72 hours) to obtain the piperidine complex.

[0035] Example 3 This embodiment is the third embodiment of the present invention. Unlike embodiment 1, the polar group in the taurine complex is an amino group, and the auxiliary components are polyethylene glycol-polylysine copolymer, lipoic acid, and methoxy polyethylene glycol. The mass ratio of taurine, modified β-cyclodextrin, and auxiliary components is 1:6:8, and the mass ratio of polyethylene glycol-polylysine copolymer, lipoic acid, and methoxy polyethylene glycol is 3:1:3.

[0036] Among them: the strong hydrophilicity of the PEG chain in polyethylene glycol-polylysine copolymer (PEG-PLL) can significantly improve the overall water solubility; the amino group (-NH2) of PLL can form "cooperative hydrogen bonds" with the amino group of cyclodextrin, reducing molecular aggregation; and the steric hindrance of the PEG chain can weaken the shielding of the cyclodextrin cavity by the amino group, which is conducive to the entry of taurine into the cavity; lipoic acid contains carboxyl group (-COOH) and sulfur group (-SH). The carboxyl group can form an acid-base neutralization effect with the amino group (-NH2), reducing the intermolecular aggregation of the amino group and improving water solubility; the reducing property of the sulfur group can protect the phenolic hydroxyl group of taurine from oxidation and maintain its structural stability; the methoxy end of methoxy polyethylene glycol (mPEG) reduces intermolecular hydrogen bonds, and the strong hydrophilicity of the PEG chain directly enhances water solubility; its flexible chain can wrap around cyclodextrin, preventing the aggregation of amino-modified cyclodextrin through steric hindrance, and does not affect the entry of taurine into the cavity.

[0037] The preparation method of the amino (-NH2) modified β-cyclodextrin includes the following steps: (1) Hydroxyl activation: Dissolve β-cyclodextrin in DMF and stir in a water bath at 60°C until completely dissolved; slowly add 2.5g TsCl (β-CD to TsCl molar ratio 1:2), then add 1mol / L NaOH solution to adjust the pH of the system to 9~10, and react at 60°C in the dark for 4 hours to obtain the p-toluenesulfonate modified β-cyclodextrin (Ts-β-CD) intermediate; (2) Amine substitution: Add excess ethylenediamine to the above reaction solution, heat to 70°C, and stir for 6 hours; (3) Purification: After the reaction is complete, the mixture is poured into anhydrous ethanol to precipitate. After standing for 2 hours, the mixture is filtered and the precipitate is collected. The precipitate is washed with anhydrous ethanol, dissolved in deionized water, and placed in a dialysis bag with a molecular weight cutoff of 3500 Da. Dialysis with deionized water is performed for 48 hours (the water is changed every 8 hours) to remove small molecule impurities. (4) Drying: After dialysis, the solution was freeze-dried to obtain amino-modified β-cyclodextrin (NH2-β-CD).

[0038] The preparation method of the taxanein complex includes the following steps: S1. Inclusion of Taxonitrile: Amine-modified β-cyclodextrin was dissolved in deionized water and stirred in a 50°C water bath until completely dissolved; then, the ethanol solution of taxonitrile was slowly added dropwise to the cyclodextrin solution in proportion, and stirred at 50°C for 2 hours (300 r / min), during which time the ethanol gradually evaporated; then, the mixture was sonicated for 30 minutes (300 W power, 50°C) to promote host-guest inclusion and form a taxonitrile-cyclodextrin inclusion complex suspension; S2. Mixing auxiliary components: Add auxiliary components to the inclusion complex solution, stir evenly, and dry to obtain the tamarindin complex, specifically: S21. Add auxiliary components: Add PEG-PLL, lipoic acid and mPEG to the piperidin-cyclodextrin inclusion complex suspension in proportion, and continue stirring at 50°C for 1 hour to obtain a mixture; S22. Homogenization and Stabilization: The mixture was transferred to a high-pressure homogenizer and homogenized three times at 20 MPa to make the system more uniformly dispersed; then stirred at room temperature for 30 minutes and allowed to cool naturally to 25°C. S23. Drying and shaping: Freeze-dry the final mixture (-50℃, vacuum degree 10Pa) to obtain the taurine complex.

[0039] Example 4 This embodiment is the fourth embodiment of the present invention. The difference from embodiment 1 is that the mass ratio of taxonomist, modified β-cyclodextrin and auxiliary components in the taxonomist complex is 1:4:9.

[0040] Example 5 This embodiment is the fifth embodiment of the present invention. The difference from embodiment 1 is that the mass ratio of taxonomist, modified β-cyclodextrin and auxiliary components in the taxonomist complex is 1:8:5.

[0041] Example 6 This embodiment is the sixth embodiment of the present invention. Unlike embodiment 1, the auxiliary component in the piperidine complex is only a hydrophilic polymer.

[0042] Example 7 This embodiment is the 7th embodiment of the present invention. Unlike embodiment 1, the auxiliary component in the piperidine complex is only an antioxidant.

[0043] Example 8 This embodiment is the 8th embodiment of the present invention. Unlike embodiment 1, the auxiliary component in the taxonomic compound is only a targeting carrier.

[0044] Example 9 This embodiment is the 9th embodiment of the present invention. Unlike embodiment 1, the auxiliary components in the piperidine complex are only hydrophilic polymers and antioxidants.

[0045] Example 10 This embodiment is the 10th embodiment of the present invention. Unlike embodiment 1, the auxiliary components in the piperidine complex are only hydrophilic polymers and targeting carriers.

[0046] Example 11 This embodiment is the 11th embodiment of the present invention. Unlike embodiment 1, the auxiliary components in the piperidine complex are only antioxidants and targeting carriers.

[0047] Comparative Example 1 This comparative example is the first comparative example of the present invention. Unlike Example 1, the β-cyclodextrin in the piperidine complex component is not modified with polar groups.

[0048] Comparative Example 2 This comparative example is the second comparative example of the present invention. Unlike Example 1, the β-cyclodextrin in the piperidine complex component is modified with a nonpolar alkyl group.

[0049] The preparation method of the methyl-modified β-cyclodextrin includes the following steps: (1) Substrate dissolution and alkaline environment adjustment. β-cyclodextrin was added to deionized water and stirred in a 45°C water bath until completely dissolved to form a transparent solution; then 10% NaOH solution was slowly added dropwise to the transparent solution to adjust the pH of the solution to 11-12 and stirring was continued for 15 min. (2) Methylation reaction. Control the reaction temperature at 30-40℃, slowly add dimethyl sulfate at a molar ratio of 5:1 to β-cyclodextrin, continuously stir during the addition, and maintain the pH at 11-12 with 10% NaOH solution; after the addition is complete, raise the temperature to 50℃ and continue the reaction for 4-6 hours; (3) Neutralization and impurity removal. After the reaction is complete, cool to room temperature and slowly add 10% HCl solution to adjust the pH to 7.0; then transfer the solution to a dialysis bag (3500 Da) and dialyze with deionized water for 24-48 hours (changing the water 3-4 times) to remove unreacted dimethyl sulfate, sodium sulfate and other small molecule impurities; (4) Purification and drying. The dialyzed solution was concentrated under reduced pressure (50℃, vacuum degree 0.08MPa) to 1 / 3 of the original volume, and 3 times the volume of methanol was added. The mixture was stirred to precipitate a white precipitate. After standing for 2 hours, the precipitate was collected by vacuum filtration and washed 2-3 times with a small amount of methanol. The precipitate was placed in a vacuum drying oven and dried at 60℃ for 8 hours to obtain methyl-modified β-cyclodextrin.

[0050] Comparative Example 3 This comparative example is the third comparative example of the present invention. Unlike Example 1, no auxiliary components were added to the fraction of the piperidine complex.

[0051] Performance testing: 1. Inclusion rate test of the taxonomic pine bark extract complex: The taxonomic pine bark extract complex was determined by HPLC. The complex was dissolved in pure water at room temperature with a mass concentration of 5%. The initial total taxonomic pine bark extract (M) was calculated by multiplying the total mass added by the amount of taxonomic pine bark extract. After complete dissolution, the mixture was centrifuged at 10,000 rpm for 30 minutes. The total mass of the centrifuged liquid was weighed, and the taxonomic pine bark extract content was determined by HPLC. The total amount of taxonomic pine bark extract encapsulated (A) was then calculated. The inclusion rate of the taxonomic pine bark extract complex was then calculated using the following formula: Inclusion rate (%) = [Total encapsulated taxine A / Initial total taxine M] × 100% The inclusion rate test results of the piperidine complexes obtained in Examples 1-11 and Comparative Examples 1-3 are shown in Table 1.

[0052] 2. Water solubility test of taxipine glycoside complex: Disperse the taxipine glycoside complex in excess in pure water at 25°C, and agitate at this temperature until dissolution equilibrium is reached (if no obvious insoluble matter is present, the amount added needs to be increased). After dissolution equilibrium is reached, filter the solution and weigh it; this is the total mass M of the solution. Use HPLC to determine the taxipine glycoside content in the solution, and then calculate the mass A of the taxipine glycoside complex. Then calculate the water solubility of the taxipine glycoside complex using the following formula: Water solubility (%, 25℃) = [Mass of soluble piperidine complex A / Total mass of solution M] × 100% The water solubility test results of the piperidine complexes obtained in Examples 1-11 and Comparative Examples 1-3 are shown in Table 1.

[0053] Table 1. Encapsulation rate and water solubility of the piperidine complex in Examples 1-11 and Comparative Examples 1-3

[0054] 3. Anti-aging function test of the piperidine complex I. Purpose and Principle of Anti-aging Function Testing This test aims to evaluate the anti-aging efficacy of Tamarind extract in Examples 1, 6, Comparative Example 1, and Comparative Example 2. To comprehensively assess the anti-aging efficacy of Tamarind extract in Examples 1, 6, Comparative Example 1, and Comparative Example 2, this invention uses the results of matrix metalloproteinase-1 (MMP-1) activity inhibition experiments as the primary evaluation criterion for anti-aging function. MMP-1 is a member of the matrix metalloproteinase (MMP) family and is a type of zinc ion-dependent proteolytic enzyme. MMPs can degrade almost all components of the extracellular matrix (ECM), including collagen and elastin. In the skin, MMP-1 mainly degrades type I and type III collagen secreted by dermal fibroblasts; these two types of collagen are the main supporting components of skin structure. When MMP-1 is overexpressed, it specifically degrades extracellular matrix components, disrupting the normal structure of collagen and elastin fibers, leading to aging symptoms such as wrinkles and decreased elasticity.

[0055] II. Anti-aging Function Testing Experimental Procedures (1) HSF cells were seeded into 96-well plates at a density of 2 × 10⁵ cells per well. The seeded 96-well plates were incubated in an incubator at 37°C and 5% CO₂ for 24 hours. The culture medium in each well was aspirated. 2 mL of H₂O₂ solution (200 μmol / L) was added to each well of the experimental group and incubated for 1 hour. After incubation, each well was washed with PBS.

[0056] (2) 200 μL of test substance culture medium of various concentrations was added to each well of the experimental group to control the concentration of active ingredients in Example 1, Example 6, Comparative Example 1 and Comparative Example 2 to be at the same level. Only 200 μL of fresh culture medium was added to each well of the blank group. The treated 96-well plate was placed in an incubator at 37°C and 5% CO2 for 24 hours.

[0057] (3) Total RNA was extracted using TRIzol reagent. cDNA was synthesized according to the instructions of the reverse transcription kit. The reaction conditions were: incubation at 37°C for 15 minutes, followed by heating at 85°C for 5 seconds. For real-time quantitative PCR, a reaction system of 20 μL was prepared, which included: 10 μL 2×ChamQ Universal SYBR qPCR Master Mix, 0.4 μL PCR Forward Primer (10 μmol / L), 0.4 μL PCR Reverse Primer (10 μmol / L), 1.0 μL cDNA template, and 8.2 μL DEPC water. The primers used were as follows: Matrix metalloproteinase-1 (MMP-1) upstream primer: 5'-GGGAAACCAGATGCTGAAACC-3'; downstream primer: 5'-TTGCAAATCTGGCGTGTAA-3'; Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal reference gene upstream primer: 5'-ATGAAGCAGCCCAGATGTGGAG-3'; downstream primer: 5'-TGGTCCACATCTGCTCTTGGCA-3'. Real-time quantitative PCR amplification conditions were: 95℃, 30 seconds, 1 cycle; 95℃, 10 seconds, 60℃, 30 seconds, 40 cycles; 95℃, 15 seconds, 60℃, 60 seconds, 95℃, 15 seconds, 1 cycle.

[0058] The anti-aging performance test results of the piperidine complexes obtained in Examples 1, 6, Comparative Example 1, and Comparative Example 2 are shown in Table 2 and... Figure 1 From Table 2, Figure 1 It can be concluded that Examples 1, 6, Comparative Example 1, and Comparative Example 2 can all effectively downregulate the activity of MMP-1. Among them, each test group has similar activity levels within the tested concentration range. At the same active ingredient concentration, the inhibition rate of each test group on MMP-1 is as follows: Example 1 > Example 6 > Comparative Example 1 > Comparative Example 2. The inhibition rates of Examples 1 and 6 are significantly better than those of Comparative Example 1 and Comparative Example 2. Among them, Example 1 has the most prominent inhibition effect and has excellent anti-aging effect.

[0059] Table 2 Effects of the test substance on MMP-1 (mean, n=3)

[0060] 4. Test on the soothing effect of the piperidine complex I. Principle of Soothing Effect Test This invention uses the expression levels of TNF-α, IL-6, and IL-1β as evaluation criteria to compare the anti-inflammatory and soothing abilities of the piperidine complexes obtained in Examples 1, 6, 1, and 2. LPS (lipopolysaccharide) is a lipid and polysaccharide complex, mainly extracted from the cell walls of Gram-negative bacteria, and possesses strong immunogenicity. LPS can stimulate skin keratinocytes to secrete pro-inflammatory cytokines (IL-1β, IL-6, etc.).

[0061] II. Test Steps (1) RNA extraction and reverse transcription polymerase chain reaction (RT-PCR) This invention employs an enzyme-linked immunosorbent assay (ELISA). To extract total RNA, cultured cells were lysed using Trizol reagent. 200 μL of chloroform was then added and vortexed, followed by incubation at room temperature for 5 minutes. The supernatant was then obtained by centrifugation at 14,000 rpm for 15 minutes at 4°C. The supernatant was mixed with isopropanol at a 1:1 ratio and centrifuged again at 14,000 rpm for 15 minutes at 4°C. RNA was reverse transcribed into cDNA using the ReverTra Ace-α kit at 42°C for 20 minutes, 99°C for 5 minutes, and then at 4°C for 5 minutes. RT-PCR reactions were performed using a StepOnePlus real-time PCR system. TaqMan probes were used for each gene, and the PCR reaction consisted of 45 cycles, each cycle including 2 minutes at 50°C, 10 minutes at 95°C, 15 seconds at 95°C, and 1 minute at 60°C.

[0062] (2) Total protein was extracted using RIPA lysis buffer. Total protein was extracted using RIPA lysis buffer. The following experimental groups were set up: blank group; LPS treatment group (5 μg / mL LPS); positive control (5 μg / mL DEX + 5 μg / mL LPS); Example 1 group (5 μg / mL, 10 μg / mL, 20 μg / mL Example 1 + 5 μg / mL LPS); Example 6 treatment group (5 μg / mL, 10 μg / mL, 20 μg / mL Example 6 + 5 μg / mL LPS); Comparative Example 1 treatment group (5 μg / mL, 10 μg / mL, 20 μg / mL Comparative Example 1 + 5 μg / mL LPS); Comparative Example 2 treatment group (5 μg / mL, 10 μg / mL, 20 μg / mL Comparative Example 2 + 5 μg / mL LPS).

[0063] (3) After determining the total protein content, each experimental group was loaded onto a 10% polyacrylamide gel and electrophoresed at 160V for 1 hour. Proteins were separated according to size and then transferred to a polyvinylidene fluoride (PVDF) membrane at room temperature for 1 hour. The PVDF membrane was blocked with 10% (w / v) skim milk powder, washed with 1×TBST buffer (50 mM Tris, 150 mM NaCl, 0.1% Tween 20, pH 7.4), and incubated with primary antibody (1:1000 dilution) at 4°C for 12 hours. After that, it was washed 3 times with 1×TBST buffer for 10 minutes each time. Then, the membrane was incubated with secondary antibody diluted with 4% skim milk powder (1:10000 dilution) for 1 hour. After washing 3 times with 1×TBST buffer again, Western ECL substrate was added to the PVDF membrane.

[0064] (4) All experimental data were analyzed using t-tests, using the mean and standard deviation of three measurements. If the p-value in the t-test was less than 0.05, statistical significance was considered. #, p<0.05; ##, p<0.01; ###, p<0.001 (calculated compared with the blank group). *, p<0.05; **, p<0.01; ***, p<0.001 (calculated compared with the induced group). The absolute difference of at least three independent measurements obtained under repeatability conditions should not exceed 20% of the arithmetic mean.

[0065] (5) Dexamethasone (5 μg / mL) was used as a positive control. At safe concentrations of the active ingredient, LPS-induced HaCaT cells were treated with Examples 1, 6, Comparative Example 1, and Comparative Example 2, and the expression levels of inflammatory cytokines and chemokines (TNF-α, IL-6, IL-1β) were detected. The test results of the piperidine complexes obtained in Examples 1, 6, Comparative Example 1, and Comparative Example 2 are shown in Table 3 and... Figures 2-4 .

[0066] As shown in Table 3 and Figure 2 As shown, compared with the blank group, the expression of IL-6 mRNA in the LPS-treated group was significantly upregulated (P < 0.001). At active ingredient concentrations of 5, 10, and 20 μg / mL, the treatment groups of Example 1, Example 6, Comparative Example 1, and Comparative Example 2 all showed varying degrees of inhibitory effects on IL-6 mRNA expression, with Example 1 showing superior performance compared to the other groups. When the active ingredient concentration of Example 1 reached 20 μg / mL, the inhibitory effect on inflammatory cytokines was superior to that of dexamethasone (DEX) at 5 μg / mL. It can be concluded that at the same active ingredient concentration, Example 1 exhibits better inhibitory effects on IL-6 mRNA expression and stronger anti-inflammatory capabilities.

[0067] As shown in Table 3 and Figure 3 As shown, compared with the blank group, IL-1β mRNA-related expression was upregulated in the LPS-treated group. At active ingredient concentrations of 5, 10, and 20 μg / mL, the experimental results of the treatment groups of Taxodiacetic acid (Examples 1, 6, Comparative Example 1, and Comparative Example 2) showed that each treatment group exhibited different degrees of inhibitory effect on IL-1β mRNA-related expression. At the same active ingredient concentration, Example 1 showed a stronger inhibitory effect on IL-1β mRNA-related expression.

[0068] As shown in Table 3 and Figure 4 As shown, compared with the blank group, the LPS treatment group upregulated the expression of TNF-α mRNA. At active ingredient concentrations of 5, 10, and 20 μg / mL, the results of the taxane treatment groups (Examples 1, 6, Comparative Example 1, and Comparative Example 2) showed that the test substance exhibited different degrees of inhibitory effect on the expression of TNF-α mRNA. At the same active ingredient concentration, Example 1 showed a stronger inhibitory effect on the expression of TNF-α mRNA, demonstrating a stronger anti-inflammatory ability.

[0069] 5. Oil-controlling effect test of piperidine complex I. Experimental Objectives and Principles This invention observes the effects of adding the piperidine complexes obtained in Examples 1, 6, Comparative Example 1, and Comparative Example 2 to the culture medium of differentiating 3T3-L1 cells on lipid accumulation and compares their lipid-controlling abilities. 3T3-L1 cells are a mouse preadipocyte cell line widely used in lipid metabolism research. Under specific differentiation conditions, these cells can differentiate into mature adipocytes and accumulate lipids. 3T3-L1 cell differentiation was induced, and intracellular lipid accumulation was monitored using Oil Red O staining. Oil Red O is a lipid-soluble dye that can selectively stain intracellular neutral lipids (such as triglycerides), thereby reflecting intracellular lipid content. Examples 1, 6, Comparative Example 1, and Comparative Example 2 have the potential to regulate lipid metabolism.

[0070] Table 3. Effects of test substances on the expression of pro-inflammatory factors (mean, n=3)

[0071] II. Experimental Procedure (1) Cell differentiation induction When 3T3-L1 cells transition from the rapid division phase to the contact inhibition phase, contact inhibition is applied for 24 hours to allow the cells to exit the growth cycle. Induction medium containing IBMX, dexamethasone, and insulin is added, and the cells are cultured for 24 hours. Then, the medium is switched to insulin-only medium, and cultured for another 24 hours. Finally, the medium is switched to standard medium without induction hormones, with the medium changed every 24 hours.

[0072] (2) Observation and staining of lipid accumulation Observe lipid droplet formation: During the induced differentiation process, observe cell morphology regularly, especially the formation of lipid droplets.

[0073] Oil Red O staining: When lipid droplets are observed, perform Oil Red O staining. The staining solution is a supersaturated solution, diluted with 1×PBS and centrifuged. Use the upper layer of staining solution with less precipitate for staining.

[0074] (3) Inhibition test At different stages of induced differentiation, the inhibitors to be tested were added, and their effects on lipid accumulation were observed. Changes in cell morphology after inhibitor addition were recorded, especially changes in lipid droplet size and number. OilRed O after staining was extracted with isopropanol, and the optical density (OD value) at 510 nm was measured using a spectrophotometer.

[0075] (4) Results Analysis The degree of lipid accumulation was quantified by measuring the area or number of lipid droplets after staining. Image processing software was used for quantification. Statistical analysis was performed on the quantification results to compare the effects of different inhibitors on lipid accumulation and the effects of inhibitors at different concentrations. #, p<0.05; ##, p<0.01; ###, p<0.001 (calculated compared with the blank group). *, p<0.05; **, p<0.01; ***, p<0.001 (calculated compared with the induced group). The oil-controlling effect test results of the piperidine complexes obtained in Examples 1, 6, Comparative Example 1, and Comparative Example 2 are shown in Table 4 and... Figure 5 .

[0076] From Table 4 and Figure 5 The results show that the effects of each test substance on lipid accumulation in macrophage-differentiated 3T3-L1 cells are as follows: at the same concentration of active ingredient, compared with Example 6, Comparative Example 1 and Comparative Example 2, Example 1 has a better inhibitory effect on lipid accumulation produced by 3T3-L1 cells, that is, Example 1 has a better oil control effect.

[0077] Table 4. Effects of test substances on lipid accumulation (mean, n=3)

[0078] The above description is merely an embodiment of the present invention and does not limit the scope of patent protection. Any non-substantial changes or substitutions made by those skilled in the art based on the present invention will still fall within the scope of patent protection.

Claims

1. A taxifolin complex, characterized in that, The complex comprises modified beta-cyclodextrin, taxifolin and auxiliary components, wherein the modified beta-cyclodextrin is modified by a polar group; the auxiliary components are selected from one or more of hydrophilic polymers, antioxidants and targeting carriers.

2. The taxifolin complex according to claim 1, characterized by The polar group is selected from amine group, sulfonyl group, carboxyl group, quaternary ammonium salt group, sugar group or polyethylene glycol chain.

3. The taxifolin complex according to claim 2, characterized by The polar group is selected from carboxyl group, amine group or sulfonyl group.

4. The taxifolin complex according to claim 2, characterized by The hydrophilic polymer is selected from polyvinylpyrrolidone, hydroxypropyl methyl cellulose, chitosan, dextran, methoxypolyethylene glycol or polyethylene glycol-polylysine copolymer.

5. The taxifolin complex according to claim 2, characterized by The antioxidant is selected from vitamin E, glutathione, tea polyphenol, coenzyme Q10 or thioctic acid.

6. The taxifolin complex of claim 2, wherein, The targeting carrier is selected from targeting peptide, folic acid, monoclonal antibody, monoclonal antibody fragment, aptamer or nanocarrier.

7. The taxifolin complex according to claim 1, characterized by The mass ratio of the taxifolin, modified beta-cyclodextrin and auxiliary components is 1:(4-8):(5-9).

8. A process for the preparation of a taxifolin complex according to any one of claims 1 to 7, characterized in that, The method comprises the following steps: S1. Inclusion of taxifolin: adding modified beta-cyclodextrin and taxifolin into a solvent in a certain proportion, stirring and reacting at 30-50℃ for 2-4 hours to form an inclusion complex suspension; S2. Mixing of auxiliary components: adding auxiliary components into the inclusion complex suspension, stirring uniformly and drying to obtain a taxifolin complex.

9. Use of the taxifolin complex of any one of claims 1-7 in the preparation of a cosmetic product.

10. Use according to claim 9, characterized in that, The cosmetic product has the effects of antioxidation, soothing, anti-aging and oil control. The cosmetic product has the effects of antioxidation, soothing, anti-aging and oil control.

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