Supramolecular bakuchiol, preparation method and composition containing supramolecular bakuchiol

Abstract

The invention provides supramolecular bakuchiol, a preparation method and a composition containing the supramolecular bakuchiol. The supramolecular bakuchiol is prepared from bakuchiol and matrine meadowfoam seed oil ionic liquid through an ultrasonic process. The composition containing the supramolecular bakuchiol further comprises a protective agent and an auxiliary material. The invention also provides an application of the supramolecular bakuchiol composition in skin care products. According to the supramolecular bakuchiol disclosed by the invention, the permeability of the bakuchiol is improved by utilizing the amphipathicity (hydrophilic and oleophylic) of the matrine meadowfoam seed oil ionic liquid, the transdermal absorption of the bakuchiol is enhanced, the bioavailability of the bakuchiol is improved, and the bakuchiol can fully play a role. The composition containing supramolecular bakuchiol has better permeability than commercially available retinol derivative products, can tighten skin and resist inflammation through multiple mechanisms, achieves better soothing and anti-aging effects, has a refreshing and pleasant skin feeling, and is easily accepted by consumers.

Description

Technical Field

[0001] The invention relates to the field of daily chemicals, and in particular to a supramolecular bakuchiol, a preparation method and a composition containing the supramolecular bakuchiol and application of the composition in skin care products. Background Art

[0002] With the improvement of living standards, people's pursuit of "beauty" is gradually increasing, and products for facial wrinkle removal and anti-aging are emerging in an endless stream. Retinoic acid (Retinoic acid) and its derivatives are an important category among them.

[0003] Retinoic acid and its derivatives include retinoic acid, retinyl esters and retinol. These substances can improve skin roughness and make the skin smooth by enhancing skin metabolism and promoting the shedding of the stratum corneum. They can also remove wrinkles and resist aging. However, retinoic acid can irritate the skin when used directly. Therefore, it often appears in the form of retinol and retinyl esters in skin care products. After the skin absorbs retinol and retinyl esters, they can slowly degrade into retinoic acid to achieve the effect of skin care. However, retinol and retinyl esters are still irritating to the skin, and some people find it difficult to build tolerance, so their application is limited.

[0004] Psoralea corylifolia, also known as Psoralea corylifolia, is the mature fruit of the leguminous plant Psoralea corylifolia. The main chemical components of Psoralea corylifolia include coumarins, terpenoids, flavonoids, etc. Bakuchiol, also known as Psoralea corylifolia, is one of the main active ingredients of Psoralea corylifolia seeds and belongs to the monoterpenoid class of substances. Bakuchiol can also regulate most of the key genes of the vitamin A signaling pathway, such as retinol binding protein CRBP, TIG1, etc. CRBP1 (retinol binding protein 1) can regulate the transport and binding of retinol, and promoting expression can activate the vitamin A signaling pathway. Compared with retinoic acid and retinol, which are prone to cause skin irritation and allergies, and are intolerant to some people, bakuchiol is better tolerated and less irritating.

[0005] However, bakuchiol is poorly soluble in water, and has poor diffusivity and permeability, resulting in low bioavailability and poor efficacy. Therefore, improving the permeability of bakuchiol is of great significance in its application. Chinese patent CN113509442A discloses a liposome containing bakuchiol and a preparation method thereof, wherein bakuchiol, lecithin and cholesterol are added to caprylic / capric triglyceride and 1,3-propylene glycol, melted to obtain an oil phase; acetylated sodium hyaluronate is added to water and dissolved to obtain an aqueous phase; the oil phase is added to the aqueous phase, and high pressure homogenization is performed to obtain a liposome containing bakuchiol. Liposomes can improve the transdermal absorption effect of the active ingredient bakuchiol, but the liposome preparation method is cumbersome and not conducive to industrial application. Therefore, there is still a lack of a bakuchiol with good permeability that can be obtained by a simple method on the market. Summary of the invention

[0006] In view of this, the present invention provides a supramolecular bakuchiol and a preparation method thereof.

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

[0008] A supramolecular bakuchiol is prepared from bakuchiol and an ionic liquid, wherein the mass ratio of the bakuchiol to the ionic liquid is 0.1-1.5:1.

[0009] Furthermore, in the supramolecular bakuchiol, the mass ratio of bakuchiol to ionic liquid is 0.8 to 1:1.

[0010] Furthermore, in the supramolecular bakuchiol, the ionic liquid is matrine and meadowfoam seed oil ionic liquid.

[0011] The bakuchiol used in the present invention is a monoterpene compound isolated from the leguminous plant psoralea corylifolia. Compared with retinol, it is mild, has low irritation to the skin, and has no photosensitivity. Scientific studies have shown that bakuchiol has the effects of oil control, anti-acne, antibacterial and anti-inflammatory, and is even better than retinol, so it is very suitable for acne patients and oily acne skin. Secondly, it can also be used to treat skin dark yellow and post-inflammatory pigmentation. Its whitening principle is to inhibit the activity of tyrosine monophenolase and diphenolase, and play a certain whitening effect. In addition, it is a fat-soluble antioxidant stronger than vitamin E, which can inhibit free radical-induced lipid peroxidation, and can also prevent ultraviolet damage to the skin, delay cell apoptosis, thereby achieving antioxidant and anti-aging effects. By reducing the decomposition of collagen and promoting the synthesis of collagen, it has also been medically proven to have good anti-aging effects. Foreign studies have shown that after using bakuchiol for 12 weeks continuously, the patient's pigmentation, light damage, skin elasticity, and wrinkle relaxation have all improved, and there is no common allergic reaction to retinol, such as dryness and peeling. Therefore, bakuchiol is an ideal substitute for vitamin A, which is less irritating and suitable for a wider range of people, and can also be used during the day. Bakuchiol is especially suitable for people with sensitive skin who need antioxidant, anti-inflammatory and antibacterial effects.

[0012] Matrine has multiple effects such as antiviral, anti-inflammatory, antibacterial, and immune system regulation. Meadowfoam seed oil contains more than 98% long-chain fatty acids with antioxidant effects. It is one of the most stable plant oils in the world and is widely used in skin care products as a cosmetic base oil. Meadowfoam seed oil has the function of preventing water loss and regenerating the skin. It is a skin metabolism activator; it changes keratin tissue, regulates the exudation of water and compounds, reabsorbs water and forms hydrates in epidermal cells to lock moisture in the skin epidermis. Matrine and meadowfoam seed oil are prepared into ionic liquids, which can play many advantages that traditional solvents cannot match. Ionic liquids are non-volatile, non-flammable, highly conductive, high in heat capacity, low in vapor pressure, stable in nature, and have good solubility in many inorganic salts and organic matter. They are widely used in electrochemistry, organic synthesis, catalysis, separation and other fields.

[0013] Bakuchiol is soluble in oil but not in water, and matrine and meadowfoam seed oil ionic liquid is a natural amphoteric solvent that is both hydrophilic and lipophilic. Using matrine and meadowfoam seed oil ionic liquid to promote the penetration of bakuchiol can effectively increase the transdermal delivery effect of bakuchiol and make it have better bioavailability.

[0014] The present invention also provides a method for preparing the supramolecular bakuchiol, comprising the following steps:

[0015] S1. Providing ionic liquid: weighing the ionic liquid and putting it into the reactor after cleaning and disinfection;

[0016] S2. Preparation of a premix: Weigh bakuchiol, keep away from light, put into a reactor containing an ionic liquid under an inert atmosphere, stir to obtain a premix;

[0017] S3. Preparation of a crude product: The premix obtained in S2 was transferred to an ultrasonic device and subjected to ultrasonic treatment to obtain a crude product;

[0018] S4. Preparation of finished product: The crude product obtained in S3 was filtered to obtain the supramolecular bakuchiol finished product;

[0019] Furthermore, in the above-mentioned preparation method, in step S2, the system temperature is controlled to be 20-45° C., the stirring speed is 200-800 rpm / min, and the stirring time is 30-60 minutes.

[0020] Furthermore, in the above-mentioned preparation method, in step S3, the system temperature is controlled to be 20-45°C, the ultrasonic power is 400-1000W, ultrasound is performed every 5-10s, each ultrasound lasts 5-10s, and the total ultrasound time is 2-4 hours.

[0021] Ultrasonic assistance is conducive to the rapid formation of supramolecular structures, and intermittent ultrasound is conducive to controlling the temperature, preventing long-term continuous ultrasound from causing the temperature to rise too high and destroying the activity of the efficacy. When the ultrasonic power is 500-700W, the degree of reaction can be better controlled to prevent excessive reaction from causing uneven local reaction.

[0022] Furthermore, in the above-mentioned preparation method, in step S4, the crude product is filtered through a 600-1000 mesh filter to obtain a finished supramolecular bakuchiol product.

[0023] Filtering with a 600-1000 mesh filter can remove impurities brought into the raw materials and introduced during the production process, making the supramolecular bakuchiol more pure, which is beneficial for subsequent storage and application.

[0024] The present invention also provides a soothing and anti-aging composition containing the supramolecular bakuchiol. In addition to the supramolecular bakuchiol, the composition also includes a protective agent and auxiliary materials;

[0025] Calculated by weight, the soothing and anti-aging composition comprises: 10 to 35 parts of supramolecular bakuchiol, 1 to 10 parts of a protective agent, and 55 to 90 parts of auxiliary materials.

[0026] Furthermore, in the above-mentioned composition, the protective agent is selected from one or more of ascorbic acid and its esters, tocopherol and its esters, and pentaerythritol tetrakis (di-tert-butyl hydroxyhydrocinnamate).

[0027] Furthermore, in the above-mentioned composition, the auxiliary material is selected from one or more of isohexadecane, cetyl ethylhexanoate, and polydimethylsiloxane.

[0028] Furthermore, the above-mentioned composition includes 30 parts by weight of supramolecular bakuchiol, 3 parts by weight of a protective agent, and 78.5 parts by weight of auxiliary materials; the protective agent includes 2 parts by weight of tocopherol acetate and 1 part by weight of pentaerythritol tetra(di-tert-butyl hydroxyhydrocinnamate); the auxiliary materials include 23.5 parts by weight of isohexadecane, 30 parts by weight of cetearyl ethylhexanoate, and 25 parts by weight of polydimethylsiloxane.

[0029] The present invention also provides an application of the soothing and anti-aging composition in skin care products.

[0030] The beneficial effects of the present invention are as follows:

[0031] (1) The supramolecular bakuchiol disclosed in the present invention uses matrine and meadowfoam seed oil ionic liquid to promote penetration, and utilizes the amphiphilicity (both hydrophilic and lipophilic) of matrine and meadowfoam seed oil ionic liquid to improve the permeability of bakuchiol, enhance its transdermal absorption, and improve the bioavailability of bakuchiol, so that bakuchiol can more fully exert its efficacy.

[0032] (2) The present invention discloses a composition comprising supramolecular bakuchiol, which has better permeability than commercially available retinol derivatives, and achieves better soothing and anti-aging effects through multiple mechanisms such as inhibiting elastase and MMP-1 enzymes, promoting fibroblast proliferation and increasing their activity, promoting collagen production, inhibiting protein degradation, and inhibiting inflammatory factors.

[0033] (3) The present invention discloses a composition comprising supramolecular bakuchiol, which has a refreshing skin feel, is not sticky or heavy, and the skin feel of the subsequent finished product is more easily accepted by consumers. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The invention is further described using the accompanying drawings, but the embodiments in the accompanying drawings do not constitute any limitation to the invention. A person skilled in the art can derive other drawings based on the following drawings without creative work.

[0035] Figure 1 is a two-dimensional nuclear magnetic resonance NOESY spectrum of supramolecular bakuchiol prepared in Example 1-1 of the present invention;

[0036] Figure 2 This is a product picture of the soothing and anti-aging composition prepared in Example 2-1 of the present invention;

[0037] FIG3 is a graph showing the inhibition rate of each sample on MMP-1 enzyme activity in performance test example 5 of the present invention, wherein Figure 3A is a graph showing the inhibition rate of the soothing and anti-aging composition prepared in Example 2-1 on the MMP-1 enzyme activity; Figure 3B This is a graph showing the inhibition rate of commercially available competitors on MMP-1 enzyme activity; Figure 3C This is a graph showing the inhibition rate of samples 2-5 on MMP-1 enzyme activity;

[0038] FIG. 4 is a graph showing the content of human type I collagen synthesized by cells after using each sample in performance test example 7 of the present invention, wherein Figure 4A This is a graph showing the content of human type I collagen synthesized by cells after using the soothing and anti-aging composition prepared in Example 2-1; Figure 4B This is a graph of the amount of human type I collagen synthesized by cells after using a commercial competitor; Figure 4C is a graph showing the content of human type I collagen synthesized by cells after using samples of comparative examples 2-5;

[0039] FIG5 is a comparison chart of the relative survival rates of cells after using each sample in performance test example 8 of the present invention, wherein Figure 5A is a comparison chart of relative cell survival rates after using the soothing and anti-aging composition prepared in Example 2-1; Figure 5B This is a comparison chart of the relative cell survival rate after using commercially available competing products; Figure 5C is a comparison chart of the relative survival rates of cells after using samples of comparative examples 2-5;

[0040] FIG. 6 is a comparison chart of the relative content of TNF-α in cells after using each sample in performance test example 9 of the present invention, wherein Fig. 6A is a comparison chart of the relative content of TNF-α in cells after using the soothing and anti-aging composition prepared in Example 2-1; Figure 6B This is a comparison chart of the relative content of TNF-α after using commercially available competing products; Figure 6C It is a comparison chart of the relative content of TNF-α in cells after using the samples of Comparative Examples 2-5. DETAILED DESCRIPTION

[0041] The experimental methods used in the following examples are conventional methods unless otherwise specified; the reagents, materials, etc. used in the following examples are commercially available products, or can be prepared by known methods unless otherwise specified. The experimental methods in the following examples without specifying specific conditions are usually carried out according to conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer.

[0042] Example 1 Preparation of supramolecular bakuchiol

[0043] S1 provides an ionic liquid: cleaning and disinfecting the reactor, adding matrine white meadowfoam seed oil ionic liquid to the reactor under a dark, inert atmosphere, turning on stirring, and controlling the temperature at 35 ° C;

[0044] S2. Preparation of premix: Keep in dark, inert atmosphere, add bakuchiol to the reactor containing matrine white meadowfoam seed oil ionic liquid, control the temperature at 35 ° C, stirring speed 600 rpm, stirring for 45 minutes to obtain a premix;

[0045] S3. Preparation of crude product: The premix obtained in S2 was transferred to an ultrasonic device with an ultrasonic power of 600 W. The premix was ultrasonically treated for 10 s every 10 s. The temperature was controlled at 35 ° C. The total ultrasonic time was 2.5 h to obtain a crude product.

[0046] S4. Preparation of finished product: The crude product obtained in S3 is filtered through an 800-mesh filter to obtain the finished supramolecular bakuchiol.

[0047] Other parameters are shown in Table 1.

[0048] Table 1 Preparation process of supramolecular bakuchiol

[0049]

[0050]

[0051] Example 2 Preparation of soothing and anti-aging composition

[0052] S1. In a light-proof condition and argon atmosphere, add isohexadecane, pentaerythritol tetra(di-tert-butyl hydroxyhydrocinnamate) ester or ascorbic acid (ester), and cetearyl ethylhexanoate into the reactor. Raise the temperature to 85° C. and stir at 600 rpm for 2 h.

[0053] S2. Cool down to 40°C, add supramolecular bakuchiol, polydimethylsiloxane and tocopherol acetate prepared in Example 1-1, and stir at 600 rpm for 30 min.

[0054] Other parameters are shown in Table 2.

[0055] Table 2 Preparation of soothing and anti-aging composition

[0056]

[0057] Comparative Examples 2-3 to 2-5 are provided, with ordinary oil base as the control:

[0058] Comparative Example 2-3: 5 g of bakuchiol was mixed with 45 g of caprylic acid / capric acid triglyceride, and stirred at 600 rpm for 30 min to obtain a 10% bakuchiol caprylic acid / capric acid triglyceride solution.

[0059] Comparative Example 2-4: 25 g of bakuchiol was mixed with 25 g of caprylic acid / capric acid triglyceride, and stirred at 600 rpm for 30 min to obtain a 50% bakuchiol caprylic acid / capric acid triglyceride solution.

[0060] Comparative Example 2-5: 10 g of the supramolecular bakuchiol prepared in Example 1-1 was mixed with 40 g of caprylic / capric triglyceride and stirred at 600 rpm for 30 min to obtain a control sample.

[0061] Performance test example

[0062] 1. Structural characterization of supramolecular bakuchiol

[0063] The sample prepared in Example 1-1 was subjected to two-dimensional nuclear magnetic resonance NOSEY test and the spectrum was analyzed.

[0064] In nuclear magnetic resonance, if two (groups) of different types of protons are close in space, irradiating one (group) of protons will enhance the signal intensity of the other (group) of protons. This phenomenon is called the nuclear Overhauser effect, or NOE effect for short. The NOESY spectrum is a two-dimensional spectrum that records all 1H-1H internuclear Ostwald effects (NOE effects) in a molecule in an experiment. The cross peaks (correlation points) of the NOESY spectrum indicate protons that are close to each other in space, that is, the information they provide is through space rather than through chemical bond interactions. Therefore, the NOESY spectrum provides very important information about the three-dimensional structure of the molecule.

[0065] Figure 1 The two-dimensional nuclear magnetic resonance NOESY spectrum of the supramolecular bakuchiol prepared in Example 1-1 clearly shows that bakuchiol and matrine white meadowfoam seed oil have related points at (H1 3.7ppm, H2 1.5ppm) and (H1 1.5ppm, H2 3.7ppm), (H1 3.7, H2 2.0ppm) and (H1 2.0, H2 3.7ppm), indicating that there is interaction between them, hydrogen bonds exist, and supramolecular is formed. That is, the preparation method of the present invention successfully prepared supramolecular bakuchiol.

[0066] 2. Test on the transdermal effect of supramolecular bakuchiol

[0067] Pig skin was used as the in vitro transdermal experimental material, and the transdermal effect of supramolecular bakuchiol was tested by Franz diffusion cell.

[0068] Select undamaged pig skin under a dissecting microscope, cut the skin of the same size, wash it once with physiological saline, and dry the surface moisture with filter paper. Franz diffusion cell, effective diffusion area is 0.36πcm 2 , the volume of the receiving pool is 15mL, the treated skin is naturally fixed between the supply pool and the receiving pool of the diffusion pool, the stratum corneum faces the supply pool, and the inner layer of the skin is just in contact with the receiving solution. Supply solution is added to the supply pool, and receiving solution is added to the receiving pool. The water bath is kept at 37°C, and the stirring speed is 300rpm. The skin is cut and extracted at specific time points (2h, 4h, 6h), and HPLC detection is performed after filtration with a 0.22μm organic membrane, and the intradermal retention is calculated in ug. The supply solution is the supramolecular bakuchiol prepared in each group of Example 1 and the 50% bakuchiol caprylic acid / capric acid triglyceride solution prepared in Comparative Examples 2-4. The receiving solution is 0.9% normal saline.

[0069] Each group of experiments was conducted three times in parallel, and SPSS 22.0 was used for data statistics. P < 0.5 was considered to be significantly different. The experimental results are shown in Table 3.

[0070] Table 3 Results of different bakuchiol in vitro transdermal experiments

[0071]

[0072] As can be seen from the data of Table 3, at the same mass concentration of bakuchiol, after skin incubation for 2h, 4h, and 6h, the bakuchiol intradermal retention of Examples 1-1 to 1-4 and Comparative Example 1-1 is 3 times higher than the bakuchiol intradermal retention of Comparative Example 2-4. The bakuchiol intradermal retention at each time point of Comparative Example 1-2 is also higher than Comparative Example 2-4, but lower than Examples 1-1 to 1-4 and Comparative Example 1-1. In Comparative Example 2-4, the solvent used is caprylic / capric triglyceride, which is a common oil and fat solvent. Experiments have shown that matrine white meadowfoam seed oil ionic liquid can play a significant penetration-promoting effect on bakuchiol compared with conventional solvents. When bakuchiol is excessive (Comparative Example 1-1), the bakuchiol intradermal retention is limited by the ratio of ionic liquids, and there is no obvious increase, and the excessive amount of active substance added has the risk of poor stability and increased irritation. However, excessively high ultrasonic power leads to uneven local reaction, affecting the transdermal performance of the prepared supramolecular bakuchiol.

[0073] 3. Transdermal effect test of soothing and anti-aging composition

[0074] Pig skin was used as the in vitro transdermal experimental material, and the transdermal effect of supramolecular bakuchiol was tested by Franz diffusion cell.

[0075] The acquisition of experimental materials, Franz diffusion cell parameters, detection time points and detection methods are the same as in “2. Test of the transdermal effect of supramolecular bakuchiol”. The supply liquid is the soothing and anti-aging composition prepared in each group of Example 2, the 10% bakuchiol caprylic acid / capric acid triglyceride solution prepared in Comparative Examples 2-3 and the commercially available competitor Granactive Retinoid (The Ordinary, purchased from Grant, the specific ingredients are 90% dimethyl isosorbide, 10% hydroxypinacol retinoic acid ester, hereinafter referred to as the commercially available competitor), and the receiving liquid is 0.9% normal saline.

[0076] Each group of experiments was conducted three times in parallel, and SPSS 22.0 was used for data statistics. P < 0.5 was considered to be significantly different. The experimental results are shown in Table 4.

[0077] Table 4 In vitro transdermal test results of different soothing and anti-aging compositions

[0078]

[0079]

[0080] It can be seen from the data in Table 4 that at the same active substance mass concentration, after 2h, 4h, and 6h of skin incubation, the intradermal retention of bakuchiol in Examples 2-1 to 2-3 is 3 times higher than that of the bakuchiol in Comparative Example 2-3, and is 9 times, 7 times, and 4 times that of the commercially available competitive products, respectively. The intradermal retention of bakuchiol at each time point in Comparative Examples 2-1 and 2-2 is also higher than that in Comparative Example 2-3 and the commercially available competitive products, but lower than that in Examples 2-1 to 2-3. Experiments have shown that the soothing and anti-aging composition prepared by the present invention still has a significant penetration-promoting effect on bakuchiol, and this penetration-promoting effect is significantly higher than that of commercially available competitive products. The lack of protective agents or excipients will affect this penetration-promoting effect.

[0081] 4. Elastase activity inhibition experiment

[0082] The soothing and anti-aging composition prepared in Example 2-1, the control sample prepared in Comparative Example 2-5 and a commercially available competitive product were used to conduct an elastase activity inhibition experiment, with the positive control being sivelestat sodium and the negative control being PBS buffer.

[0083] Set up sample tube (A1), sample background tube (A2), enzyme activity control group (A3) and substrate blank group (A4). For each sample tube (A1) of the tested concentration, three parallel tubes need to be set up. Add 25 μL of sample solution to the quality tube (A1) and the quality background tube (A2), respectively, and add the same volume of PBS buffer to the enzyme activity control group (A3) and the substrate blank group (A4) to replace the sample. Add 225 μL of elastase solution to each tube in turn, mix and heat in a 37°C water bath for 5 minutes, add 250 μL of substrate solution to the sample tube (A1) and the enzyme activity control group (A3), add the same volume of 10% DMSO solution to the sample background tube (A2) and the substrate blank group (A4), mix and heat in a 37°C water bath for 30 minutes, and measure the absorbance at a wavelength of 382 nm.

[0084]

[0085] The experimental results are shown in Table 5 below.

[0086] Table 5 Elastase inhibition rate of different samples

[0087]

[0088] Remark:

[0089] (1) When the concentration of the commercially available competitor Granactive Retinoid sample is greater than 1%, the reaction system becomes milky after centrifugation, which has a significant impact on the detection.

[0090] (2) The data in the table are mean ± relative deviation.

[0091] (3) Statistical method: The t-test method was used for analysis, and the significance level α = 0.05. Compared with NC, P ≥ 0.05 indicates no statistical difference; 0.01 < P < 0.05 indicates significant difference; P < 0.01 indicates very significant difference, and P < 0.001 indicates extremely significant difference.

[0092] Elastin, as an important structural protein in skin composition, is degraded during skin aging due to the increased content of matrix metalloproteinases such as elastase, resulting in loss of skin elasticity. Elastase (human leukocyte elastase, HLE) is a destructive enzyme present in the body that can hydrolyze certain connective tissue components such as elastin, proteoglycans, and certain types of collagen. Elastase can specifically hydrolyze substrates, and the absorbance value of the released p-nitroaniline (pNA) at 382 nm was measured by spectrophotometry to calculate the enzyme activity and inhibition rate.

[0093] As shown in the above table, the soothing and anti-aging composition prepared in Example 2-1 could not calculate the IC50 for the inhibition of elastase activity, but had an inhibitory effect on elastase, indicating that the sample had a firming effect and could effectively resist aging. The sample in Comparative Example 2-5 could not calculate the IC50 for the inhibition of elastase activity, and the inhibition rate was lower than that of the sample in Example 2-1 at the same concentration. The commercially available competitor could not calculate the IC50 for the inhibition of elastase activity and P ≥ 0.05, showing no inhibitory effect on elastase and no firming effect.

[0094] 5. MMP-1 Enzyme Activity Inhibition Experiment

[0095] The soothing and anti-aging composition prepared in Example 2-1, the control sample prepared in Comparative Example 2-5, and the commercially available competitor were used for the elastase activity inhibition experiment. The positive control was o-phenanthroline, and the negative control was PBS buffer.

[0096] A sample group (A1), a sample background group (A2), an enzyme activity control group (A0), and a substrate blank group (A4) were established. For each test concentration of the sample, three parallels of the sample group (A1) were set up. The MMP-1 enzyme solution (2200 μg / μL) and APMA (30 mmol / L) were mixed at a volume ratio of 10:1 and incubated at 37 °C for 90 min to obtain the activated MMP-1 solution. 50 μL of the fluorescent substrate DQ-gelatin solution and 40 μL of the activated MMP-1 solution were added to the sample group (A1) and the enzyme activity control group (A0) respectively. 10 μL of the sample solution was added to the sample group (A1) and the sample background group (A2). The sample background group (A2) and the substrate blank group (A4) were made up to 100 μL with HEPES buffer. Immediately, the 96-well plate was placed in a fluorescence microplate reader and allowed to stand in the dark at 37 °C for 30 min. The fluorescence intensity was measured at an excitation wavelength of 460 nm and an emission wavelength of 520 nm.

[0097]

[0098] The experimental results are shown in Table 6 below.

[0099] Table 6 Inhibition rate of MMP-1 enzyme activity of different samples

[0100]

[0101]

[0102] Remarks:

[0103] (1) The data in the table are mean ± relative deviation.

[0104] (2) Statistical method: The t-test method was used for analysis, and the test level α = 0.05. Compared with NC, P ≥ 0.05 indicates no statistical difference; 0.01 < P < 0.05 indicates significant difference; P < 0.01 indicates very significant difference, and P < 0.001 indicates extremely significant difference.

[0105] According to the matrix metalloproteinase aging theory, external factors cause the activity of matrix metalloproteinase-1 (MMP-1) to increase, which over-degrades the extracellular matrix, destroys the skin structure, and leads to the formation of wrinkles. If a certain functional ingredient can effectively inhibit the activity of matrix metalloproteinase-1, it can reduce the destruction and loss of the extracellular matrix, prevent the formation of wrinkles, and achieve the purpose of anti-wrinkle. The fluorescent substrate DQ-gelatin emits fluorescence after being hydrolyzed by MMP-1 enzyme. Raw materials with MMP-1 enzyme activity inhibitory effect can reduce the hydrolysis amount of the fluorescent substrate DQ-gelatin, thereby reducing the fluorescence intensity. By detecting the change of the fluorescence intensity of the reaction system with a fluorescence microplate reader, the inhibitory effect of the raw material on the activity of MMP-1 enzyme can be evaluated.

[0106] As shown in Table 6 above and Figures 3A to 3C as shown, when the soothing anti-aging composition prepared in Example 2-1 is at a volume concentration of 1% to 10%, it has an inhibitory effect on MMP-1 enzyme. The sample of Comparative Example 2-5 has an inhibitory effect on MMP-1 enzyme at a volume concentration of 1% to 10%. However, the inhibition rate is lower than that of the sample of Example 2-1 at the same volume concentration. The commercially available competitor has an inhibitory effect on MMP-1 enzyme at a volume concentration of 5%. Therefore, the soothing anti-aging composition prepared in Example 2-1, the sample of Comparative Example 2-5, and the commercially available competitor all have anti-wrinkle effects, and the soothing anti-aging composition prepared in Example 2-1 is superior to the sample of Comparative Example 2-5 and the commercially available competitor.

[0107] 6. Determination of the activity of MMPS in human skin fibroblasts

[0108] An experiment on the determination of the activity of MMPS in human skin fibroblasts was carried out using the soothing anti-aging composition prepared in Example 2-1, the control sample prepared in Comparative Example 2-5, and the commercially available competitor.

[0109] The experimental scheme is as shown in Table 7 below.

[0110] Table 7 Experimental scheme

[0111]

[0112] The test results are as shown in Tables 8-1 to 8-3 below.

[0113] Table 8-1 Test results of the activity of MMPS in human skin fibroblasts of the sample of Example 2-1

[0114]

[0115] Table 8-2 Test results of the activity of MMPS in human skin fibroblasts of the sample of Comparative Example 2-5

[0116]

[0117] Table 8-3 Test results of the activity of MMPS in human skin fibroblasts of the commercially available competitor

[0118]

[0119] Note: Statistical method: Analysis was carried out using the t-test method, and the test level α = 0.05. Compared with NC, P≥0.05 indicates no statistical difference; 0.01 < P < 0.05 indicates a significant difference; P < 0.01 indicates a very significant difference, and P < 0.001 indicates an extremely significant difference. 0.01 < P < 0.05 is expressed as *, P < 0.01 is expressed as **, and P < 0.001 is expressed as ***.

[0120] One of the main causes of skin aging is the change in the structure of the dermis. There are many reasons for the change in the structure of the dermis. Some external factors, such as ultraviolet radiation, activate the matrix metalloproteinase (MMPs) in the body, which can lead to excessive degradation of collagen and elastin in the dermis that support the skin structure, resulting in wrinkles and inelasticity. MMPs are a group of proteolytic enzymes containing active Zn. According to the different structures of the catalytic region, they can be divided into collagenase, gelatinase, matrix lysin, stromelysin, and membrane-type MMPs. The most representative one is collagenase, which can decompose the triple helix structure of type I, II, and I collagen fibers. Fluorescence resonance energy transfer (FRET) is a non-radiative energy transition that transfers the energy of the donor excited state to the acceptor excited state through the electric dipole interaction between molecules. This analytical method has the advantages of rapidity and sensitivity. The test substance is added to the human skin fibroblast culture medium for cultivation, and the MMPs activity in the cell supernatant is detected by FRET technology to analyze whether the test substance can inhibit the activity of MMPs and evaluate the anti-wrinkle effect of the sample.

[0121] As shown in Tables 8-1 to 8-3 above, compared with the negative control group, the soothing and anti-aging composition prepared in Example 2-1 had a significant decrease in the relative content of MMPs at the test concentrations of 0.12 mg / mL and 0.06 mg / mL (P<0.05), and the inhibition rates were 29.90% and 47.88%, respectively. This indicates that the soothing and anti-aging composition prepared in Example 2-1 has the effect of inhibiting the activity of MMPs at the concentrations of 0.12 mg / mL and 0.06 mg / mL, achieving an anti-wrinkle effect.

[0122] Compared with the negative control group, the relative content of MMPs in the commercially available competing products at the test concentrations of 0.12 mg / mL and 0.06 mg / mL decreased significantly (P<0.05), and the inhibition rates were 26.91% and 26.03%, respectively. This indicates that the commercially available competing products have the effect of inhibiting the activity of MMPs at the concentrations of 0.12 mg / mL and 0.06 mg / mL, achieving anti-wrinkle effects.

[0123] Compared with the negative control group, the relative content of MMPs in the samples of comparative examples 2-5 at the test concentrations of 0.12 mg / mL and 0.06 mg / mL decreased significantly (P<0.05), and the inhibition rates were 25.74% and 28.42%, respectively. This indicates that the samples of comparative examples 2-5 at the concentrations of 0.12 mg / mL and 0.06 mg / mL have the effect of inhibiting the activity of MMPs and achieving anti-wrinkle effect.

[0124] In summary, the soothing anti-aging composition prepared in Example 2-1, the samples of Comparative Example 2-5, and the commercially available competitors can all inhibit the activity of MMPs. Moreover, at the same concentration, the action ability of the soothing anti-aging composition prepared in Example 2-1 is superior to that of the commercially available competitors and the samples of Comparative Example 2-5, demonstrating that the anti-wrinkle effect of the soothing anti-aging composition prepared in Example 2-1 is superior to that of the commercially available competitors and the samples of Comparative Example 2-5.

[0125] Determination of Human Type I Collagen Content in Example 7

[0126] An experiment on the determination of human type I collagen content was carried out using the soothing anti-aging composition prepared in Example 2-1, the control samples prepared in Comparative Example 2-5, and the commercially available competitors.

[0127] The experimental scheme is as shown in Table 9 below.

[0128] Table 9 Experimental Scheme

[0129]

[0130]

[0131] The experimental results are as shown in Tables 10-1 to 10-3 below.

[0132] Table 10-1 Detection Results of Human Type I Collagen Content in the Sample of Example 2-1

[0133]

[0134] Table 10-2 Detection Results of Human Type I Collagen Content in the Commercially Available Competitors

[0135]

[0136] Table 10-3 Detection Results of Human Type I Collagen Content in the Samples of Comparative Example 2-5

[0137]

[0138] Note: Statistical method: Analysis was carried out using the t-test method, and the test level α = 0.05. Compared with the BC group, P-value < 0.001 is expressed as . Compared with the NC group, P ≥ 0.05 indicates no statistical difference; 0.01 < P < 0.05 indicates a significant difference; P < 0.01 indicates a very significant difference, and P < 0.001 indicates an extremely significant difference. 0.01 < P < 0.05 is expressed as *, P < 0.01 is expressed as **, and P < 0.001 is expressed as ***.

[0139] Type I collagen is one of the main components of the extracellular matrix of the dermis. It is synthesized by dermal fibroblasts and secreted outside the cells. Under the action of terminal procollagen peptidase, the terminal peptides are separated and polymerized to form collagen fibers. Therefore, the firming and anti-wrinkle effect of the sample to be tested can be evaluated by the content of type I collagen secreted by fibroblasts after administration.

[0140] As shown in Tables 10-1 to 10-3 and Figures 4A to 4C As shown, compared with the negative control group, the content of human type I collagen in the positive control group increased significantly (P<0.05), indicating that this experiment is effective. Compared with the negative control group, the soothing and anti-aging composition prepared in Example 2-1 has a significant increase in the content of human type I collagen at the test concentrations of 0.030%, 0.015%, and 0.010% (P<0.05), and the upregulation rates are 142.84%, 126.04%, and 75.11%, respectively. This means that the soothing and anti-aging composition prepared in Example 2-1 has the ability to promote the synthesis of human type I collagen at concentrations of 0.030%, 0.015%, and 0.010%, achieving anti-wrinkle and firming effects.

[0141] Compared with the negative control group, the human type I collagen content of the commercially available competing products did not increase significantly at the test concentrations of 0.030%, 0.015%, and 0.010%, which means that the commercially available competing products have no ability to promote the synthesis of human type I collagen at concentrations of 0.30%, 0.015%, and 0.010%.

[0142] Compared with the negative control group, the human type I collagen content of the comparative example 2-5 samples at the test concentration of 0.030% was significantly increased (P<0.05), and the up-regulation rate was 23.42%; at the test concentrations of 0.015% and 0.010%, the human type I collagen content did not increase significantly, which means that the comparative example 2-5 samples only have the ability to promote the synthesis of human type I collagen at a high concentration of 0.30%, achieving anti-wrinkle and firming effects, but have no ability to promote the synthesis of human type I collagen at the concentrations of 0.015% and 0.010%.

[0143] Comprehensive analysis shows that the ability to promote the synthesis of human type I collagen is as follows: the soothing and anti-aging composition prepared in Example 2-1 > the sample in Comparative Example 2-5 > the commercially available competitor. That is, the soothing and anti-aging composition prepared in Example 2-1 can effectively promote the synthesis of human type I collagen and achieve the effect of firming and anti-wrinkle.

[0144] 8. Human skin fibroblast proliferation test

[0145] The soothing and anti-aging composition prepared in Example 2-1, the control sample prepared in Comparative Example 2-5, and a commercially available competitive product were used to conduct a human skin fibroblast proliferation promotion test experiment.

[0146] The experimental plan is shown in Table 11 below.

[0147] Table 11 Test Plan

[0148]

[0149] The experimental results are shown in Tables 12-1 to 12-3 below.

[0150] Table 12-1 Proliferation Promotion Test Results of Example 2-1 Samples on Human Skin Fibroblasts

[0151]

[0152] Table 12-2 Proliferation Promotion Test Results of Commercially Available Competing Products on Human Skin Fibroblasts

[0153]

[0154]

[0155] Table 12-3 Proliferation Promotion Test Results of Example 2-5 Samples on Human Skin Fibroblasts

[0156]

[0157] Note: Statistical method: Analysis was performed using the t-test method, and the significance level α = 0.05. Compared with the BC group, P-value < 0.001 is denoted as . Compared with the NC group, P ≥ 0.05 indicates no statistical difference; 0.01 < P < 0.05 indicates a significant difference; P < 0.01 indicates a very significant difference; P < 0.001 indicates an extremely significant difference. 0.01 < P < 0.05 is denoted as *, P < 0.01 is denoted as **, and P < 0.001 is denoted as ***.

[0158] Fibroblasts are the main cell components of loose connective tissue and are differentiated from mesenchymal cells during embryonic development. Fibroblasts are relatively large, with clear outlines, mostly spindle-shaped or star-shaped flat structures with protrusions. Their cell nuclei are regular oval shapes, and the nucleoli are large and obvious. Fibroblasts are highly active in functional activities. Their cytoplasm is weakly basophilic, with obvious protein synthesis and secretion activities. Under certain conditions, they can be transformed into fibrocyte mutually. Fibroblasts play a very important role in the repair of cell degeneration, necrosis, tissue defects of different degrees, and bone trauma. In December 2013, researchers at King's College London in the UK found that fibroblasts have different types, and their unique properties can help repair damaged skin and reduce the impact of aging on the skin.

[0159] As shown in Tables 12-1 to 12-3 above and Figures 5A to 5CAs shown, compared with the negative control group, the number of cells in the positive control group increased significantly (P<0.05), indicating that this experiment is effective. Compared with the negative control group, the soothing and anti-aging composition prepared in Example 2-1 did not increase significantly in number of cells at the test concentrations of 0.015% and 0.010%, indicating that the soothing and anti-aging composition prepared in Example 2-1 had no proliferation-promoting effect at the concentrations of 0.015% and 0.010%; at the test concentration of 0.030%, the number of cells increased significantly (P<0.05), and the proliferation rate was 11.52%, which means that the soothing and anti-aging composition prepared in Example 2-1 has a proliferation-promoting effect at a concentration of 0.03%, achieving a repairing effect.

[0160] Compared with the negative control group, the number of cells did not increase significantly at the test concentrations of 0.030%, 0.015%, and 0.010% for the commercially available competitor, which means that the commercially available competitor has no proliferation-promoting effect at the concentrations of 0.030%, 0.015%, and 0.010%.

[0161] Compared with the negative control group, the cell number of comparative example 2-5 samples at the test concentrations of 0.030%, 0.015%, and 0.010% did not increase significantly, which means that comparative example 2-5 samples had no proliferation promoting effect at the concentrations of 0.030%, 0.015%, and 0.010%.

[0162] Comprehensive analysis shows that the ability of the soothing and anti-aging composition prepared in Example 2-1 to promote the proliferation of human skin fibroblasts is greater than that of the sample in Comparative Example 2-5 and the commercially available competing products. That is, the soothing and anti-aging composition prepared in Example 2-1 can effectively promote the proliferation of human skin fibroblasts, thereby repairing damaged skin and reducing skin aging.

[0163] 9. Determination of TNF-α inflammatory factor content

[0164] The soothing and anti-aging composition prepared in Example 2-1, the control sample prepared in Comparative Example 2-5, and a commercially available competitive product were used to perform an experiment to determine the content of TNF-α inflammatory factor.

[0165] The experimental scheme is shown in Table 13 below.

[0166] Table 13 Experimental plan

[0167]

[0168]

[0169] The experimental results are shown in Tables 14-1 to 14-3 below.

[0170] Table 14-1 Results of determination of TNF-α inflammatory factor content in samples of Example 2-1

[0171]

[0172] Table 14-2 Results of the determination of TNF-α inflammatory factor content in commercially available competing products

[0173]

[0174] Table 14-3 Results of the determination of TNF-α inflammatory factor content in the samples of Comparative Examples 2-5

[0175]

[0176] Note: Statistical method: Analysis was performed using the t-test method, and the significance level α = 0.05. Compared with the BC group, P-value < 0.001 is expressed as . Compared with the NC group, P ≥ 0.05 indicates no statistical difference; 0.01 < P < 0.05 indicates a significant difference; P < 0.01 indicates a very significant difference; P < 0.001 indicates an extremely significant difference. 0.01 < P < 0.05 is expressed as *, P < 0.01 is expressed as **, and P < 0.001 is expressed as ***.

[0177] LPS-induced mouse macrophage RAW 264.7 is a classic cell model for studying inflammatory factors. By comparing the differences in the TNF-α content of mouse macrophage RAW 264.7 after modeling control and administration of the test substance, the effect of the test substance on inhibiting TNF-α secretion was evaluated.

[0178] As shown in Tables 14-1 to 14-3 above and Figures 6A to 6C as shown, compared with the model group, the soothing anti-aging composition prepared in Example 2-1 showed a significant decrease in TNF-α content (P < 0.05) at test concentrations of 0.015%, 0.010%, and 0.005%, and the inhibition rates were 18.37%, 19.59%, and 20.16%, respectively. This indicates that the soothing anti-aging composition prepared in Example 2-1 can inhibit TNF-α secretion at concentrations of 0.015%, 0.010%, and 0.005%, achieving the effects of reducing inflammation and soothing the skin.

[0179] Compared with the model group, the commercially available competing products did not show a significant decrease in TNF-α content at test concentrations of 0.010% and 0.005%, indicating that the commercially available competing products have no effect on inhibiting TNF-α secretion at concentrations of 0.010% and 0.005%; at a test concentration of 0.015%, the TNF-α content decreased significantly (P < 0.05), and the inhibition rate was 9.72%, indicating that the commercially available competing products can inhibit TNF-α secretion at a high concentration of 0.015%, achieving the effects of reducing inflammation and soothing the skin.

[0180] Compared with the modeling group, the TNF-α content of the comparative example 2-5 samples at the test concentrations of 0.010% and 0.005% was not significantly reduced, which means that the comparative example 2-5 samples had no inhibitory effect on TNF-α secretion at concentrations of 0.010% and 0.005%; at the test concentration of 0.015%, the TNF-α content was significantly reduced (P<0.05), and the inhibition rate was 2.68%, which means that the comparative example 2-5 samples at a high concentration of 0.015% can inhibit TNF-α secretion, thereby achieving the effect of reducing inflammation and soothing the skin.

[0181] In summary, the soothing and anti-aging composition prepared in Example 2-1 is better than the commercially available competitive products and the samples of Comparative Example 2-5 in inhibiting TNF-α secretion, that is, the soothing and anti-aging composition prepared in Example 2-1 has better effects of reducing inflammation and soothing the skin.

[0182] 10. Questionnaire survey of volunteers on skin feel of raw materials

[0183] The soothing and anti-aging composition prepared in Example 2-1, the control sample prepared in Comparative Example 2-5, and a commercially available competitive product were used to perform an experiment to determine the content of TNF-α inflammatory factor.

[0184] 30 volunteers were recruited, and three areas were divided on the inner side of the left arm. 1 mL of different samples were dropped on each area, and spread evenly with the right fingertips. Ten minutes later, the volunteers filled in the questionnaire. The questionnaire design is shown in Table 15.

[0185] Table 15 Questionnaire design

[0186]

[0187] After the experiment, questionnaires were collected to obtain the average score of each indicator for each raw material.

[0188] The experimental results are shown in Table 16 below.

[0189] Table 16 Summary results of the raw material skin feel questionnaire,

[0190] Index samples Example 2-1 Sample Comparative Example 2-5 Samples Competitive products on the market Freshness 4.83 3.97 4.53 Absorbance 4.77 4.03 4.46 Overall skin feel 4.87 3.90 4.53

[0191] As shown in Table 16 above, in the skin feel test, the scores for freshness, absorbency and overall skin feel are all Example 2-1 sample > commercially available competitor > Comparative Example 2-5 sample. It can be seen that the soothing and anti-aging composition prepared in Example 2-1 has a better skin feel, and the subsequent finished products prepared using it are more easily accepted by consumers.

[0192] Although the embodiments of the present application have been shown and described above, it is understood that the above embodiments are exemplary and cannot be construed as limitations on the present application. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present application.

Claims

1. A supramolecular bakuchiol prepared from bakuchiol and an ionic liquid, characterized in that: The mass ratio of the bakuchiol to the ionic liquid is 0.1 to 1.5:

1.

2. The supramolecular bakuchiol according to claim 1, characterized in that The mass ratio of the bakuchiol to the ionic liquid is 0.8 to 1:

1.

3. The supramolecular bakuchiol according to claim 1 or 2, characterized in that The ionic liquid is matrine and meadowfoam seed oil ionic liquid.

4. A method for preparing the supramolecular bakuchiol according to any of the above items, comprising the following steps: S1. Providing ionic liquid: weighing the ionic liquid and putting it into the reactor after cleaning and disinfection; S2. Preparation of a premix: Weigh bakuchiol, keep away from light, put into a reactor containing an ionic liquid under an inert atmosphere, stir to obtain a premix; S3. Preparation of a crude product: The premix obtained in S2 was transferred to an ultrasonic device and subjected to ultrasonic treatment to obtain a crude product; S4. Prepare finished product: Filter the crude product obtained in S3 to obtain the finished supramolecular bakuchiol product.

5. The preparation method according to claim 4, characterized in that: In step S2, the system temperature is controlled to be 20-45° C., the stirring speed is 200-800 rpm / min, and the stirring time is 30-60 minutes.

6. The preparation method according to claim 4, characterized in that: In step S3, the system temperature is controlled to be 20-45° C., the ultrasonic power is 400-1000 W, and the ultrasonic treatment is performed every 5-10 seconds. Each ultrasonic treatment lasts for 5-10 seconds, and the total ultrasonic treatment lasts for 2-4 hours.

7. The preparation method according to claim 4, characterized in that: In the step S4, the crude product is filtered through a 600-1000 mesh filter to obtain the finished supramolecular bakuchiol product.

8. A soothing and anti-aging composition comprising the supramolecular bakuchiol prepared by any one of the above items, characterized in that: The composition also includes a protective agent and auxiliary materials; Calculated by weight, the soothing and anti-aging composition comprises: 10 to 35 parts of supramolecular bakuchiol, 1 to 10 parts of a protective agent, and 55 to 90 parts of auxiliary materials.

9. The composition according to claim 8, characterized in that The protective agent is selected from one or more of ascorbic acid and its esters, tocopherol and its esters, and pentaerythritol tetrakis (di-tert-butyl hydroxyhydrocinnamate).

10. The composition according to claim 8, characterized in that The auxiliary material is selected from one or more of isohexadecane, cetyl ethylhexanoate and polydimethylsiloxane.

11. The composition according to any one of claims 8 to 10, characterized in that The invention comprises the following components in parts by weight: 30 parts by weight of supramolecular bakuchiol, 3 parts by weight of a protective agent, and 78.5 parts by weight of auxiliary materials; the protective agent comprises 2 parts by weight of tocopherol acetate and 1 part by weight of pentaerythritol tetra(di-tert-butyl hydroxyhydrocinnamate); The auxiliary materials include 23.5 parts by weight of isohexadecane, 30 parts by weight of cetearyl ethylhexanoate, and 25 parts by weight of polydimethylsiloxane.

12. Use of any of the above-mentioned soothing and anti-aging compositions in skin care products.

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