Composition with biological rhythm regulating function, its preparation method and application

Abstract

The application provides a composition with biological rhythm adjusting function and a preparation method and application thereof, and relates to the technical field of cosmetics.The composition provided by the application comprises glycyrrhizic acid, glabridin, p-hydroxyacetophenone, butanediol, 1,2-hexanediol and hydroxypropyl cyclodextrin, wherein the glycyrrhizic acid, the glabridin and the hydroxypropyl cyclodextrin form a self-assembly system.The composition has good stability, can effectively regulate biological rhythm gene expression through the synergistic cooperation of the glycyrrhizic acid, the glabridin and the hydroxypropyl cyclodextrin, treat rhythm disorder related diseases, relieve joint swelling caused by acute gout with hyperuricemia, has good antioxidant property and tyrosinase activity inhibiting effect, and shows a good application prospect in improving biological rhythm disorder related skin problems and metabolic diseases.

Description

Technical Field

[0001] This invention relates to the field of cosmetic technology, and in particular to a composition having the function of regulating biological rhythms, its preparation method, and its application. Background Technology

[0002] In the human body, the 24-hour circadian rhythm oscillates in almost every organ and every type of cell. This biological clock system precisely regulates cellular metabolism and physiological activities through a "molecular clock" composed of core clock genes (such as Clock, Bmal1, Per, Cry, etc.) to maintain synchronization between the body and the external environment. Studies have found that circadian rhythm disorders are associated with a variety of diseases. (1) The connection mechanism between sleep-deprivation-induced muscle soreness and biological rhythm Existing research confirms that disruption of the biological circadian rhythm is the root cause of skin damage caused by staying up late. During a normal physiological cycle, the skin exhibits a clear "daytime defense, nighttime repair" characteristic: high expression of the Per gene during the day promotes thickening of the stratum corneum and secretion of antioxidant enzymes to resist external stressors such as ultraviolet rays; at night, Clock and Bmal1 dominate, initiating DNA repair and cell regeneration processes. However, the abnormal light signals accompanying staying up late (especially blue light exposure) interfere with the transcription cycle of the Per gene, leading to the failure of skin defense mechanisms, accumulation of reactive oxygen species (ROS) and inflammatory factors, and severely compressing the nighttime repair window. This rhythm disorder directly manifests as typical symptoms of "staying up late skin," mainly manifested as dull and yellowish skin tone, rough and lackluster skin texture, often accompanied by facial swelling, sagging, and increased fine lines due to microcirculatory disturbances. Furthermore, the biological rhythm disruption induced by staying up late exacerbates skin inflammation, leading to redness, sensitivity, frequent acne and closed comedones. Combined with insufficient blood supply around the eyes resulting in pigmented dark circles and dryness, this creates an extremely tired, haggard, and lifeless appearance.

[0003] (2) The extensive links between biological rhythms and other diseases Besides skin problems, circadian rhythm disorders are also a key contributing factor to many serious diseases. Studies have shown that gout, acute myocardial infarction, type 2 diabetes, Alzheimer's disease, and ulcerative colitis are all closely related to circadian rhythm disturbances, highlighting the central role of circadian rhythm regulation in the prevention and treatment of cross-system diseases.

[0004] Studies have found that glycyrrhizin and glycyrrhizic acid, as natural active ingredients, have been used in the fields of anti-inflammation and anti-oxidation. However, no studies have yet revealed their efficacy in regulating biological rhythms and improving rhythm disorder-related symptoms, and there are no reports of their self-assembly systems being used in this field.

[0005] In view of this, the present invention is hereby proposed. Summary of the Invention

[0006] The first objective of this invention is to provide a composition having the function of regulating biological rhythms, so as to solve the above-mentioned technical problems.

[0007] A second objective of the present invention is to provide a method for preparing the above-described composition.

[0008] A third objective of the present invention is to provide the use of the above-described composition in the preparation of a medicament for treating diseases related to rhythm disorders.

[0009] A fourth objective of this invention is to provide the use of the above-described composition in the preparation of antioxidant products.

[0010] A fifth objective of the present invention is to provide the use of the above-described composition in the preparation of a medicament for treating joint swelling caused by acute gout with hyperuricemia.

[0011] A sixth object of the present invention is to provide the use of the above composition in the preparation of tyrosinase activity inhibitors.

[0012] The seventh objective of this invention is to provide a cosmetic product.

[0013] To achieve the above objectives, the following technical solution is adopted: In a first aspect, the present invention provides a composition having the function of regulating biological rhythms, comprising, by weight percentage: 0.3%-1.0% glycyrrhizic acid, 0.1%-3.2% glycyrrhizin, 0.3%-0.7% p-hydroxyacetophenone, 15%-25% butanediol, 1.5%-2.5% 1,2-hexanediol, 10%-25% hydroxypropyl cyclodextrin, with the balance being water; The glycyrrhizic acid, glycyrrhizin, and hydroxypropyl cyclodextrin form a self-assembling system.

[0014] Secondly, the present invention provides a method for preparing the above-mentioned composition having the function of regulating biological rhythms, comprising the following steps: A mixed solution was obtained by heating and stirring p-hydroxyacetophenone, butanediol, and 1,2-hexanediol at 60-70°C. Then, glycyrrhizic acid and glycyrrhizin were added to carry out self-assembly. After that, the mixed solution was added to an aqueous solution of hydroxypropyl cyclodextrin and mixed to prepare a composition with the function of regulating biological rhythm.

[0015] Thirdly, the present invention provides the use of the above-mentioned composition having the function of regulating biological rhythms in the preparation of a medicament for treating diseases related to rhythm disorders.

[0016] As a further technical solution, the composition having the function of regulating biological rhythms treats rhythm disorder-related diseases by upregulating the expression level of the rhythm gene PER1.

[0017] As a further technical solution, the rhythm disorder-related diseases include sleep deprivation muscle strain or gout caused by rhythm disorder.

[0018] Fourthly, the present invention provides the application of the above-mentioned composition having the function of regulating biological rhythms in the preparation of antioxidant products.

[0019] Fifthly, the present invention provides the use of the above-mentioned composition having the function of regulating biological rhythms in the preparation of a medicament for treating joint swelling caused by acute gout with hyperuricemia.

[0020] In a sixth aspect, the present invention provides the use of the above-described composition having the function of regulating biological rhythms in the preparation of tyrosinase activity inhibitors.

[0021] In a seventh aspect, the present invention provides a cosmetic comprising the above-described composition having the function of regulating biological rhythms.

[0022] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a composition with circadian rhythm regulation function, comprising glycyrrhizic acid, glycyrrhizin, p-hydroxyacetophenone, butylene glycol, 1,2-hexanediol, and hydroxypropyl cyclodextrin, wherein glycyrrhizic acid, glycyrrhizin, and hydroxypropyl cyclodextrin form a self-assembly system. This composition exhibits good stability and, through the synergistic effect of glycyrrhizic acid, glycyrrhizin, and hydroxypropyl cyclodextrin, can effectively regulate the expression of circadian rhythm genes, treat circadian rhythm-related diseases, alleviate joint swelling caused by acute gout with hyperuricemia, and possesses good antioxidant and tyrosinase-inhibiting effects. It shows promising application prospects in improving circadian rhythm-related skin problems and metabolic diseases. Attached Figure Description

[0023] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0024] Figure 1 The degree of ankle swelling in each group of rats; Figure 2 The inhibition rate of tyrosinase by each test sample is shown. Detailed Implementation

[0025] The embodiments and examples of the present invention will be described in detail below. However, those skilled in the art will understand that the following embodiments and examples are for illustrative purposes only and should not be considered as limiting the scope of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Unless otherwise specified, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0026] In a first aspect, the present invention provides a composition having the function of regulating biological rhythms, comprising, by weight percentage: 0.3%-1.0% glycyrrhizic acid, 0.1%-3.2% glycyrrhizin, 0.3%-0.7% p-hydroxyacetophenone, 15%-25% butanediol, 1.5%-2.5% 1,2-hexanediol, 10%-25% hydroxypropyl cyclodextrin, with the balance being water; The glycyrrhizic acid, glycyrrhizin and hydroxypropyl cyclodextrin form a self-assembly system (i.e., glycyrrhizic acid, glycyrrhizin and hydroxypropyl cyclodextrin self-assemble).

[0027] The composition provided by this invention comprises glycyrrhizic acid, glycyrrhizin, p-hydroxyacetophenone, butylene glycol, 1,2-hexanediol, and hydroxypropyl cyclodextrin, wherein glycyrrhizic acid, glycyrrhizin, and hydroxypropyl cyclodextrin self-assemble to form vesicles. This composition exhibits good stability and, through the synergistic effect of glycyrrhizic acid, glycyrrhizin, and hydroxypropyl cyclodextrin, can effectively regulate the expression of circadian rhythm genes, treat circadian rhythm-related diseases, and alleviate joint swelling caused by acute gout with hyperuricemia. It also possesses good antioxidant and tyrosinase-inhibiting effects, showing promising application prospects in improving circadian rhythm-related skin problems and metabolic diseases.

[0028] Furthermore, it should be noted that the glycyrrhizin in this invention can be replaced with other whitening, antioxidant, anti-wrinkle and firming, repairing, or soothing and antipruritic ingredients. For example, it can be at least one of 4-butylresorcinol, phenylethylresorcinol, tanshinone extract, cryptotanshinone, paeonol, licorice extract, glycyrrhizin chalcone A, ceramide, centella asiatica extract, asiaticoside, scutellaria baicalensis extract, baicalin, baicalein, resveratrol, dalbergia odorifera extract, pterostilbene, coenzyme Q10, psoralen, silymarin, retinol, tocopherol and its analogues, azelaic acid, and salicylic acid. Glycyrrhizic acid can be replaced with glycyrrhizic acid derivatives, such as dipotassium glycyrrhizate, glycyrrhetinic acid, and monoammonium glycyrrhizate.

[0029] In some alternative embodiments, the composition having the function of regulating biological rhythms comprises, by weight percentage: 0.5% glycyrrhizic acid, 0.1% glycyrrhizin, 0.5% p-hydroxyacetophenone, 20% butanediol, 2% 1,2-hexanediol, 20% hydroxypropyl cyclodextrin, and the balance being water.

[0030] Secondly, the present invention provides a method for preparing the above-mentioned composition having the function of regulating biological rhythms, comprising the following steps: A mixed solution was obtained by heating and stirring p-hydroxyacetophenone, butanediol, and 1,2-hexanediol at 60-70°C. Then, glycyrrhizic acid and glycyrrhizin were added to carry out self-assembly. After that, the mixed solution was added to an aqueous solution of hydroxypropyl cyclodextrin and mixed to prepare a composition with the function of regulating biological rhythm.

[0031] The preparation method is simple and convenient, and the prepared composition has good stability. The prepared composition has the effects of regulating the expression of biological rhythm genes, treating diseases related to rhythm disorder, relieving joint swelling caused by acute gout with hyperuricemia, good antioxidant activity, and inhibiting tyrosinase activity.

[0032] Thirdly, the present invention provides the use of the above-mentioned composition having the function of regulating biological rhythms in the preparation of a medicament for treating diseases related to rhythm disorders.

[0033] The inventors have discovered that the composition provided by this invention has the effect of upregulating the expression level of the rhythm gene PER1, and can therefore be used to treat rhythm disorder-related diseases (such as night sweats or gout caused by rhythm disorder).

[0034] Fourthly, the present invention provides the application of the above-mentioned composition having the function of regulating biological rhythms in the preparation of antioxidant products.

[0035] The inventors have discovered that the composition provided by this invention has excellent free radical scavenging ability and can be used in the preparation of antioxidant products.

[0036] Fifthly, the present invention provides the use of the above-mentioned composition having the function of regulating biological rhythms in the preparation of a medicament for treating joint swelling caused by acute gout with hyperuricemia.

[0037] The inventors have discovered that the composition provided by this invention can relieve joint swelling caused by acute gout accompanied by hyperuricemia.

[0038] In a sixth aspect, the present invention provides the use of the above-described composition having the function of regulating biological rhythms in the preparation of tyrosinase activity inhibitors.

[0039] The inventors have discovered that the composition provided by this invention has the effect of inhibiting tyrosinase activity and can be used to prepare tyrosinase activity inhibitors.

[0040] In a seventh aspect, the present invention provides a cosmetic comprising the above-described composition having the function of regulating biological rhythms.

[0041] This cosmetic product includes the composition of the present invention and has all the beneficial effects of the composition of the present invention.

[0042] The present invention will be further illustrated below with specific embodiments and comparative examples. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.

[0043] Example 1 A composition, by weight percentage, comprises: 0.5% glycyrrhizic acid, 0.1% glycyrrhizin, 20% hydroxypropyl cyclodextrin, 0.5% p-hydroxyacetophenone, 20% butanediol, 2% 1,2-hexanediol, and the balance being water. The preparation method is as follows: S1. Weigh p-hydroxyacetophenone, butanediol and 1,2-hexanediol, and dissolve them by heating at 65°C. This solution is denoted as A1.

[0044] S2. Weigh out glycyrrhizic acid and glycyrrhizin, add them to A1, and perform self-assembly at 65°C. This mixture is denoted as A2.

[0045] S3. Weigh out hydroxypropyl cyclodextrin and the remaining water, and dissolve them by heating at 65°C. This solution is denoted as B.

[0046] S4. Slowly add solution A2 to solution B while stirring continuously until thoroughly mixed. Store the resulting product away from light at 4℃–8℃.

[0047] Example 2 A composition, by weight percentage, comprises: 0.5% glycyrrhizic acid, 2% glycyrrhizin, 20% hydroxypropyl cyclodextrin, 0.5% p-hydroxyacetophenone, 20% butanediol, 2% 1,2-hexanediol, with the balance being water. The preparation method is the same as in Example 1.

[0048] Example 3 A composition, by weight percentage, comprises: 0.5% glycyrrhizic acid, 3.2% glycyrrhizin, 20% hydroxypropyl cyclodextrin, 0.5% p-hydroxyacetophenone, 20% butanediol, 2% 1,2-hexanediol, and the balance being water. The preparation method is the same as in Example 1.

[0049] Comparative Example 1 A glycyrrhizic acid blank composition, comprising, by weight percentage: 0.5% glycyrrhizic acid, 0.5% p-hydroxyacetophenone, 20% butanediol, 2% 1,2-hexanediol, and the balance being water. The preparation method is as follows: S1. Weigh p-hydroxyacetophenone, butanediol and 1,2-hexanediol, and dissolve them by heating at 65°C. This solution is denoted as A1.

[0050] S2. Weigh out glycyrrhizic acid and add it to A1. Perform self-assembly at 65°C and denote it as A2.

[0051] S3. Slowly add water to A2 while stirring continuously until well mixed. Store in the dark at 4℃–8℃.

[0052] Comparative Example 2 A blank composition of hydroxypropyl cyclodextrin, comprising, by weight percentage: 20% hydroxypropyl cyclodextrin, 0.5% p-hydroxyacetophenone, 20% butanediol, 2% 1,2-hexanediol, and the balance being water. The preparation method is as follows: S1. Weigh p-hydroxyacetophenone, butanediol and 1,2-hexanediol, and dissolve them by heating at 65°C. This solution is denoted as A.

[0053] S2. Weigh out hydroxypropyl cyclodextrin and the remaining water, and dissolve them by heating at 65°C. This solution is denoted as B.

[0054] S3. Slowly add solution A to solution B while stirring continuously until thoroughly mixed. Store the resulting product away from light at 4℃–8℃.

[0055] Comparative Example 3 A glycyrrhizic acid-hydroxypropyl cyclodextrin blank composition comprises: 0.5% glycyrrhizic acid, 20% hydroxypropyl cyclodextrin, 0.5% p-hydroxyacetophenone, 20% butanediol, 2% 1,2-hexanediol, and the balance being water. The preparation method is as follows: S1. Weigh p-hydroxyacetophenone, butanediol and 1,2-hexanediol, and dissolve them by heating at 65°C. This solution is denoted as A1.

[0056] S2. Weigh out glycyrrhizic acid, add it to A1, and self-assemble it at 65℃. This is denoted as A2.

[0057] S3. Weigh out hydroxypropyl cyclodextrin and add the remaining amount of water. Heat at 65°C to dissolve, and record as B.

[0058] S4. Slowly add solution A2 to solution B while stirring continuously until well mixed. Store the resulting product away from light at 4℃-8℃.

[0059] Comparative Example 4 A composition, by weight percentage, comprises: 0.5% glycyrrhizic acid, 0.1% glycyrrhizin, 0.5% p-hydroxyacetophenone, 20% butanediol, 2% 1,2-hexanediol, and the balance being water. The preparation method is as follows: S1. Weigh p-hydroxyacetophenone, butanediol and 1,2-hexanediol, and dissolve them by heating at 65°C. This solution is denoted as A1.

[0060] S2. Weigh out glycyrrhizic acid and glycyrrhizin and add them to A1. Perform self-assembly at 65°C and denote it as A2.

[0061] S3. Slowly add water to A2 while stirring continuously until well mixed. Store in the dark at 4℃–8℃.

[0062] Comparative Example 5 A composition, by weight percentage, comprises: 0.1% glycyrrhizin, 20% hydroxypropyl cyclodextrin, 0.5% p-hydroxyacetophenone, 20% butanediol, 2% 1,2-hexanediol, and the balance being water. The preparation method is as follows: S1. Weigh p-hydroxyacetophenone, butanediol and 1,2-hexanediol, and dissolve them by heating at 65°C. This solution is denoted as A1.

[0063] S2. Weigh out glycyrrhizin and add it to A1. Dissolve the dissolved substance by heating at 65°C. This solution is then labeled A2.

[0064] S3. Weigh out hydroxypropyl cyclodextrin and the remaining water, and dissolve them by heating at 65°C. This solution is denoted as B.

[0065] S4. Slowly add solution A2 to solution B while stirring continuously until thoroughly mixed. Store the resulting product away from light at 4℃–8℃.

[0066] Comparative Example 6 A composition, by weight percentage, comprises: 0.5% glycyrrhizic acid, 2% glycyrrhizin, 0.5% p-hydroxyacetophenone, 20% butanediol, 2% 1,2-hexanediol, and the balance being water. The preparation method is the same as Comparative Example 4.

[0067] Comparative Example 7 A composition, by weight percentage, comprises: 2% glycyrrhizin, 20% hydroxypropyl cyclodextrin, 0.5% p-hydroxyacetophenone, 20% butanediol, 2% 1,2-hexanediol, and the balance being water. The preparation method is the same as Comparative Example 5.

[0068] Comparative Example 8 A composition, by weight percentage, comprises: 0.5% glycyrrhizic acid, 3.2% glycyrrhizin, 0.5% p-hydroxyacetophenone, 20% butanediol, 2% 1,2-hexanediol, and the balance being water. The preparation method is the same as Comparative Example 4.

[0069] Comparative Example 9 A composition, by weight percentage, comprises: 3.2% glycyrrhizin, 20% hydroxypropyl cyclodextrin, 0.5% p-hydroxyacetophenone, 20% butanediol, 2% 1,2-hexanediol, and the balance being water. The preparation method is the same as Comparative Example 5.

[0070] Comparative Example 10 A solution of glycyrrhizin is prepared as follows: Dilute the glycyrrhizin powder with ethanol to a concentration of 0.1%.

[0071] Comparative Example 11 A simple physical mixture of glycyrrhizic acid and glycyrrhizin, comprising: 0.5% glycyrrhizic acid and 0.1% glycyrrhizin, with the balance being ethanol; the preparation method is as follows: Glycyrrhizic acid and glycyrrhizin were diluted to 0.5% and 0.1% with ethanol, respectively.

[0072] Comparative Example 12 A solution of glycyrrhizin is prepared as follows: Dilute the glycyrrhizin powder with ethanol to the appropriate 2%.

[0073] Comparative Example 13 A simple physical mixture of glycyrrhizic acid and glycyrrhizin, comprising: 0.5% glycyrrhizic acid and 2% glycyrrhizin, with the balance being ethanol; the preparation method is as follows: Glycyrrhizic acid and glycyrrhizin were diluted to 0.5% and 2% with ethanol, respectively.

[0074] Experimental Example 1: Performance Testing (Particle Size Testing) 1. Testing Method The particle size of the samples from the examples or comparative examples was determined by passing them through a 0.22 μm filter membrane.

[0075] 2. Test Results As shown in Table 1.

[0076] Table 1 Particle size results

[0077] (1) Limited self-assembly capability of single components Comparative Examples 1-3 show that glycyrrhizic acid and hydroxypropyl cyclodextrin each possess self-assembly capabilities individually. However, the blank self-assembled system formed by self-assembly alone has a relatively large particle size (>120 nm), resulting in limited self-assembly efficiency. Although the mixture of the two exhibits a certain synergistic shrinkage effect (reducing the particle size to around 88 nm), it is still far from the ideal carrier (<50 nm), limiting the self-assembly density and drug loading potential.

[0078] (2) Binary systems cannot simultaneously address both particle size and drug loading. Comparative Examples 4, 6, and 8 show that glycyrrhizic acid alone has extremely low carrying capacity for glycyrrhizin—it can self-assemble at 0.1%, but once the concentration exceeds 2%, it directly demulsifies or fails to form. This indicates that glycyrrhizic acid itself has very limited hydrophobic cavities, and it alone cannot support slightly higher concentrations of active ingredients.

[0079] Comparative Examples 5, 7, and 9 show that the self-assembly ability of hydroxypropyl cyclodextrin and glycyrrhizin is improved, but it only forms a typical cyclodextrin inclusion complex with a stable particle size of about 84-85 nm. It does not change significantly with the increase of glycyrrhizin concentration, but it can never reach the ideal self-assembly size.

[0080] (3) The ternary co-assembly achieves the synergy of "1+1+1>3".

[0081] ① The particle size is significantly reduced: from 88 nm in the blank to 15~25 nm, reaching the ideal particle size range for transdermal absorption.

[0082] ② The drug loading capacity is greatly improved: even when the amount of glycyrrhizin is increased to 3.2%, it can still stably form a self-assembled system.

[0083] ③ The synergistic mechanism is made explicit: Glycyrrhizic acid provides a self-assembly framework, and hydroxypropyl cyclodextrin further confines and stabilizes the hydrophobic core. The combined effect of the three can significantly improve the compactness of the self-assembly system and the feasibility of extremely small particle size.

[0084] Experimental Example 2: The Effect of Test Samples on Biological Rhythms Test Example 1: Testing the effects of various samples on genes related to biological rhythms 1. Testing Method B16 cells were cultured after inoculation, and the test sample (the combination of the examples and comparative examples, at a concentration of 1.5%) was added. After 2 days of incubation, total mRNA was extracted from the cells, reverse transcribed into cDNA, and then amplified. The PCR reaction was performed on a real-time quantitative PCR system.

[0085] 2. Test Results (1) Effects of the examples and their comparative examples on the expression level of the circadian rhythm gene PER1 Table 2. Expression levels of the PER1 gene, a biological rhythm gene, in each sample.

[0086] The results analysis shows that: (1) The blank self-assembly system (vector) itself does not have rhythm regulation function: the expression levels of Comparative Example 1 (glycyrrhizic acid blank self-assembly system) and Comparative Example 3 (glycyrrhizic acid + cyclodextrin blank self-assembly system) are even lower than 1, with no statistical difference (NS) compared with the control group. This indicates that the simple glycyrrhizic acid self-assembly system or the system co-assembled with hydroxypropyl cyclodextrin not only did not upregulate PER1, but was at a low expression baseline.

[0087] (2) Comparative Example 10 (glycyrrhizin solution) showed only a slight increase in values ​​compared with the control group, with no significant difference (NS).

[0088] (3) Comparative Example 11 (a simple physical mixture of glycyrrhizic acid and glycyrrhizin) significantly upregulated PER1 expression. P<0.05 This indicates that although glycyrrhizic acid and glycyrrhizin alone are not very effective, they can produce a positive regulatory effect when used together.

[0089] (4) Comparative Example 4 (glycyrrhizic acid-glaucamine self-assembly system) showed a highly significant expression-promoting effect (P<0.01), which was significantly better than that of the physical mixture and each individual component. The results indicate that glycyrrhizic acid and glaucamine alone have limited regulatory capacity on PER1, and their effect on improving circadian rhythm-related diseases (such as muscle fatigue caused by staying up late and gout) may be weak. The physical mixture has shown advantages in combination, and the glycyrrhizic acid-glaucamine self-assembly system further significantly enhances the regulatory effect. This confirms that there is a synergistic positive feedback mechanism between glaucamine and glycyrrhizic acid, and that the self-assembly system can amplify this effect; it has potential improvement effects on problems such as muscle fatigue caused by staying up late and gout caused by circadian rhythm disorders.

[0090] (5) Compared with the control group, the first group showed a highly significant expression-promoting effect. P＜0.0001 Its effect is significantly better than that of a single component ( P＜0.01 This study confirms that the glycyrrhizic acid-hydroxypropyl cyclodextrin-glycyrrhizin ternary assembly system can synergistically regulate circadian rhythms and has potential improvement effects on problems such as muscle soreness and gout caused by circadian rhythm disorders. Combined with the performance test results, the ternary assembly exhibits a synergistic effect. Under the condition of the same content of the active ingredient glycyrrhizin (0.0015%), Example 1 (glycyrrhizic acid-hydroxypropyl cyclodextrin-glycyrrhizin ternary assembly system) significantly enhanced PER1 gene expression compared to Comparative Example 4 (glycyrrhizic acid-glycyrrhizin self-assembled system). Compared to Comparative Example 4, PER1 expression increased by approximately 34%, a difference that was statistically significant. P<0.5 This indicates that the ternary assembly system not only helps to form smaller and more uniform self-assembled systems, but also further enhances the synergistic effect of the three components in regulating circadian rhythm genes, and has the potential to improve problems such as muscle soreness and gout caused by circadian rhythm disorders.

[0091] Test Example 2: Testing the effect of each sample on free radicals 1. Testing Method Weigh 7.884 mg of 1,1-diphenyl-2-trinitrophenylhydrazine powder and dilute to 100 mL with ethanol. Shake well to obtain a 0.2 mmol DPPH solution. Using a pipette, pipette 500 μL of a 1% concentration sample solution (diluted 100 times with water to the composition of the example or comparative example) into a plastic centrifuge tube, add 500 μL of DPPH solution, shake well, and protect from light for 30 min. Use ethanol as a blank control. Transfer 200 μL to each well of a 96-well plate and measure the absorbance at 517 nm. Perform three parallel experiments. The specific calculation formula is as follows: DPPH clearance rate = (1 - D1 / D2) × 100%.

[0092] In the formula, D1 is the absorbance of the test sample or positive control; D2 is the absorbance of the blank control.

[0093] 2. Test Results Table 3. Effects of each sample on free radicals

[0094] Table 3 data analysis shows the limitations of the single-component / blank self-assembled system: Comparative Examples 1 and 2 showed a DPPH radical scavenging rate of <30%; the system formed by the self-assembly of glycyrrhizic acid and hydroxypropyl cyclodextrin showed improved DPPH radical scavenging ability, but the effect was still small. Comparative Example 10 (glycyrrhizin) showed some scavenging ability, but the scavenging rate was less than 30%.

[0095] Enhancement and limitations of physical mixing: Comparative Example 11 (a simple physical mixture of glycyrrhizic acid and glycyrrhizin) showed a clearance rate of 30.72%, which was slightly higher than that of glycyrrhizin alone, but the improvement was limited.

[0096] Example 1 (glycyrrhizic acid-hydroxypropyl cyclodextrin-glaucamine self-assembled system) showed a DPPH radical scavenging rate >30%, significantly superior to physical mixtures (Comparative Example 11), single components (Comparative Examples 1, 2, 3, 10), and binary assembled systems (Comparative Examples 4, 5) of the same concentration. This confirms a significant synergistic DPPH radical scavenging effect among the glycyrrhizic acid-hydroxypropyl cyclodextrin-glaucamine self-assembled system. This self-assembled structure not only improves the stability and dispersibility of the active ingredient but also effectively enhances its free radical scavenging ability.

[0097] Test Example 3: Testing the effects of various samples on an animal model of acute gout with hyperuricemia. 1. Testing Method (1) Establishment of a rat model of acute gout with hyperuricemia After acclimatization, male SD rats, except for the normal control group, were given an acute hyperuricemia model via intraperitoneal injection of potassium oxonate emulsion (25 mg / mL, 10 mL / kg) and oral gavage with hypoxanthine suspension (100 mg / mL, 10 mL / kg). After successful model establishment, the rats' right hind legs were straightened, and 50 μL of sodium urate solution (50 mg / mL) was injected into the ankle joint to induce acute gout. The normal control group rats were injected with the same volume of 0.9% saline solution using the same method.

[0098] (2) Effects of each test sample on the "rat model of acute gout with hyperuricemia" After acclimatization, male SD rats were randomly divided into 7 groups of 6 rats each. The grouping is shown in Table 4. After successfully inducing an acute gout model with hyperuricemia 1 h according to the modeling method in (1), rats in the NC (normal group) and MC (model group) groups were given 4 mL of physiological saline (0.9%) by gavage, while the PC group (positive drug group) was treated with allopurinol (40 mg / kg). Each rat in the other groups was given 4 mL of the drug by gavage (the drug was a combination of the example or comparative example diluted 100 times with physiological saline, i.e., the drug concentration was 1%).

[0099] Table 4 Grouping of animals in acute gout with hyperuricemia model

[0100] Note: Except for the normal group, all other groups were tested using model rats.

[0101] 2. Test Results Depend on Figure 1 It can be seen that, compared with the normal group (NC), the swelling degree of rats in the acute gout with hyperuricemia model group (MC) was significantly different, and the model was successfully established; Compared with the model group (MC), Example 2 showed a significant difference. Example 2 had a better effect on joint swelling caused by acute gout with hyperuricemia, significantly better than Comparative Example 13 (simple physical mixture of the same concentration), the glycyrrhizic acid-hydroxypropyl cyclodextrin blank self-assembled system (Comparative Example 3), and glycyrrhizin (Comparative Example 12). This confirms that glycyrrhizin and the glycyrrhizic acid-hydroxypropyl cyclodextrin blank self-assembled system have a synergistic effect, and the self-assembled structure can further enhance its effect on joint swelling caused by acute gout with hyperuricemia.

[0102] Test Example 4: Testing the effects of various samples on skin damaged by lack of sleep Typical clinical manifestations of "skin dullness due to lack of sleep" include dull, yellowish skin tone and worsened dark circles. Melanin production is a key step leading to dull skin tone, with tyrosinase and tyrosinase-related proteins (TRP-1 and TRP-2) playing a central regulatory role in melanin synthesis. Therefore, inhibiting tyrosinase activity is one of the main strategies for developing melanin production inhibitors.

[0103] 1. Testing Method Test the effect of each sample on tyrosinase After B16 cells were seeded and cultured, the concentration of the sample to be tested (1.5%) was added. After 2 days of treatment, the cells were harvested. After 2 days of treatment, the cell lysate was collected and reacted with levodopa (10 mmol / L) at 37℃ for 40 minutes. The absorbance value at a wavelength of 492 nm was measured using an ELISA reader.

[0104] 2. Test Results Depend on Figure 2 It can be seen that: Comparative Example 1 (glycyrrhizic acid blank self-assembly system) has an inhibition rate of less than 5% on tyrosinase and has a poor effect on improving skin dullness caused by staying up late.

[0105] Both Comparative Example 10 (glabridin) and Comparative Example 11 (a simple physical mixture of glycyrrhizic acid and glabridin) showed an inhibition rate of approximately 30%, indicating a certain improvement effect on skin dullness caused by staying up late. However, there was no significant difference between Comparative Example 11 and Comparative Example 10 (P ≥ 0.05), suggesting that the simple physical mixture of glycyrrhizic acid and glabridin cannot produce a strong synergistic inhibitory effect on tyrosinase.

[0106] In contrast, Example 1 (glycyrrhizic acid + 0.1% glycyrrhizin + hydroxypropyl cyclodextrin self-assembled system) showed an inhibition rate of up to about 80%, which was significantly better than the physical mixture and each individual component (P<0.0001).

[0107] The above results indicate that glycyrrhizic acid alone has limited inhibitory activity against tyrosinase; glycyrrhizin possesses certain tyrosinase inhibitory activity and can alleviate skin dullness caused by circadian rhythm disorders or staying up late to some extent; simple physical mixing of the two did not show synergistic effects and failed to further enhance the inhibitory effect; while the ternary assembly of Example 1 (glycyrrhizic acid + 0.1% glycyrrhizin + hydroxypropyl cyclodextrin self-assembly system) can achieve synergistic inhibition of tyrosinase.

[0108] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A composition having the function of regulating biological rhythms, characterized in that, By weight percentage, it includes: 0.3%-1.0% glycyrrhizic acid, 0.1%-3.2% glycyrrhizin, 0.3%-0.7% p-hydroxyacetophenone, 15%-25% butanediol, 1.5%-2.5% 1,2-hexanediol, 10%-25% hydroxypropyl cyclodextrin, with the balance being water; The glycyrrhizic acid, glycyrrhizin, and hydroxypropyl cyclodextrin form a self-assembling system.

2. The method for preparing the composition with circadian rhythm regulation function according to claim 1, characterized in that, Includes the following steps: A mixed solution was obtained by heating and stirring p-hydroxyacetophenone, butanediol, and 1,2-hexanediol at 60-70°C. Then, glycyrrhizic acid and glycyrrhizin were added to carry out self-assembly. After that, the mixed solution was added to an aqueous solution of hydroxypropyl cyclodextrin and mixed to prepare a composition with the function of regulating biological rhythm.

3. The use of the composition of claim 1, which has the function of regulating biological rhythms, in the preparation of a medicament for treating diseases related to rhythm disorders.

4. The application according to claim 3, characterized in that, The composition having the function of regulating biological rhythms treats rhythm disorder-related diseases by upregulating the expression level of the rhythm gene PER1.

5. The application according to claim 3, characterized in that, The rhythm disorder-related diseases include sleep deprivation-related muscle strain or gout caused by rhythm disorders.

6. The use of the composition with circadian rhythm regulation function as described in claim 1 in the preparation of antioxidant products.

7. The use of the composition of claim 1, which has the function of regulating biological rhythms, in the preparation of a medicament for treating joint swelling caused by acute gout with hyperuricemia.

8. The use of the composition of claim 1, which has the function of regulating biological rhythms, in the preparation of tyrosinase activity inhibitors.

9. A cosmetic product, characterized in that, The composition comprising the function of regulating biological rhythms as described in claim 1.

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