Compositions and their use in improving stress and enhancing the effects of RAR agonists

Abstract

The present application discloses a composition comprising: (i) a compound of formula (I) or a precursor thereof; and (ii) glabridin or a derivative thereof. The present application also discloses the use of the composition in improving stress and related results, including in homeostasis / anti-inflammation; and in RAR agonist potentiation, and further in preparing skin care formulations. The composition disclosed in the present application, comprising the compound of formula (I) or a precursor thereof and glabridin or a derivative thereof, breaks through the technical limitations of skin care of the two components alone, wherein the compound of formula (I) or a precursor thereof and glabridin or a derivative thereof produce unexpected synergies after compounding, and achieve effect improvement in cell stress and its results including homeostasis / anti-inflammation, such as mitochondrial homeostasis / anti-inflammation, and skin homeostasis, skin anti-inflammation, skin moisturizing, skin soothing repair, skin whitening and other core skin care effects, far exceeding the effect performance of the two components used alone.

Description

Technical Field

[0001] This invention relates to the field of composition technology, and more specifically to a composition and its application in improving stress and enhancing the effects of RAR agonists. Background Technology

[0002] Under normal physiological conditions, the body relies on the coordinated efforts of multiple systems to maintain homeostasis. Various acute and chronic stress stimuli continuously disturb neuroendocrine regulatory pathways, and once the body's compensatory threshold is exceeded, homeostasis imbalance is triggered. Homeostasis imbalance can relieve the negative inhibition of the immune system, activate the innate immune inflammatory pathway, continuously secrete a variety of pro-inflammatory cytokines, and induce systemic low-grade chronic inflammation. Conversely, the continuous accumulation of inflammatory mediators can in turn interfere with the function of the stress regulation axis, aggravate the body's homeostasis disorder, and form a vicious cycle of stress-homeostasis imbalance-chronic inflammation.

[0003] Skin care is a health and beauty field that receives a great deal of attention from the public. Consumers' demand for skin care ingredients has shifted from single-function to multi-dimensional comprehensive effects such as anti-inflammation, moisturizing, repair, and whitening. Natural, gentle, and highly effective active skin care ingredients have become the core direction of research and application.

[0004] RAR agonists (including retinoids) regulate skin homeostasis processes such as keratinocyte differentiation, epidermal renewal, collagen synthesis, MMP expression, and pigment metabolism by activating the transcriptional function of the RAR / RXR heterodimer at the RARE site. The most abundant RAR and RXR subtypes in the skin are RXRα and RARγ. RAR agonists are the most well-supported class of active ingredients in photoaging treatment, acne treatment, and skin renewal. However, RAR agonists are less effective in inflamed skin.

[0005] Currently, existing technologies attribute the poor efficacy of RAR agonists in inflamed skin to poor irritation tolerance or penetration / stability issues. However, at the molecular level, even if RAR agonists successfully reach target cells and bind to RAR, sustained activation of NF-κB suppresses RAR function from the transcriptional output. Therefore, increasing the concentration of RAR agonists or improving their delivery cannot resolve this bottleneck. On the other hand, existing technologies have attempted to enhance the efficacy of RAR agonists by incorporating anti-inflammatory agents into RAR agonist formulations to reduce irritation; however, this does not address the genetic issue.

[0006] Urolithiasis A is a natural metabolite of ellagic acid produced in the human intestine. As a natural skin care active ingredient, its core application advantage lies in natural anti-aging, making it an important choice for anti-aging and repair skin care.

[0007] Glycyrrhizin is a natural active ingredient extracted from licorice root. It is known in the industry as "whitening gold" because of its gentle whitening effect. It can regulate skin melanin-related metabolism with low irritation and is a natural ingredient with great application value in whitening skin care. It is widely used in various gentle whitening skin care products.

[0008] In practical skincare applications, both urolithin and glycyrrhizin have significant limitations when used alone: ​​both are single-function skincare ingredients, with urolithin focusing on anti-aging and glycyrrhizin focusing on gentle whitening, making it difficult to simultaneously meet consumers' comprehensive skincare needs for anti-inflammation, moisturizing, soothing and repairing, and whitening; moreover, both the anti-aging effects of urolithin and the gentle whitening effects of glycyrrhizin require long-term and continuous conditioning to show results, and their effects are slow when used alone, failing to meet consumers' demand for highly effective skincare.

[0009] Furthermore, under conditions of chronic inflammation or oxidative stress, the inhibitory depth of either drug alone on the NF-κB pathway is insufficient, and they cannot effectively block the formation of the inhibitory complex between p65 and RAR, resulting in a decrease in the transcriptional efficiency of the RAR / RXR pathway, and the attenuation of RAR agonist efficacy cannot be reversed or inhibited.

[0010] Therefore, there is an urgent need for a natural active ingredient composition that can achieve stabilization and anti-inflammation at the cellular level, synergistic effects (such as skin care), and enhance the transcriptional efficiency of RAR agonists. Summary of the Invention

[0011] The technical problem to be solved by the present invention is to provide a composition in which urolithin and glycyrrhizin overcome the technical limitations of using the two components alone. When combined, they produce an unexpected synergistic effect, which improves stress and its related outcomes, such as homeostasis / anti-inflammation (cellular homeostasis / anti-inflammation), especially in skin homeostasis, skin anti-inflammation, skin moisturizing, soothing and repair, whitening and other effects, and far exceeds the efficacy of using the two components alone.

[0012] The technical problem to be solved by the present invention also includes providing a composition that unexpectedly exhibits a significant synergistic effect in enhancing the efficacy of RAR agonists.

[0013] To solve the above-mentioned technical problems, the present invention provides the following technical solution: A first aspect of the present invention provides a composition comprising or consisting of the following components: (i) A compound of formula (I) or a precursor thereof;

[0014] (ii) Glycyrrhizin or its derivatives; in: A, B, C, and D are each independently selected from H and OH; W, X, and Y are each independently selected from H and OH; and Z is selected from H and OH.

[0015] A second aspect of the present invention provides a product comprising the aforementioned composition.

[0016] A third aspect of the present invention provides a formulation comprising the aforementioned composition.

[0017] A fourth aspect of the present invention provides a formulation comprising: (1) A first formulation containing a compound of formula (I) or a precursor thereof;

[0018] as well as (2) A second preparation containing glycyrrhizin or its derivatives; in: A, B, C, and D are each independently selected from H and OH; W, X, and Y are each independently selected from H and OH; and Z is selected from H and OH.

[0019] The fifth aspect of the invention provides the use of the said composition, the said product or formulation, or the said combination of formulations in improving stress and its related outcomes such as homeostasis / anti-inflammation.

[0020] The sixth aspect of the present invention provides the use of the said composition, the said product or formulation, or the combination of said formulations in the preparation of skin care formulations.

[0021] The seventh aspect of the present invention provides the use of the said composition, the said product or formulation, or the combination of said formulations in skin care.

[0022] The eighth aspect of the present invention provides the use of the said composition, the said product or formulation, or the combination of said formulations in the preparation of formulations for enhancing the effect of RAR agonists.

[0023] The ninth aspect of the present invention provides the use of the said composition, the said product or formulation, or the combination of said formulations in enhancing the effect of RAR agonists.

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention combines the compound of formula (I) or its precursor with glycyrrhizin or its derivatives for the first time and found that it has a significant synergistic effect in improving stress and related outcomes, such as cell homeostasis / anti-inflammation, especially mitochondrial homeostasis / anti-inflammation.

[0025] 2. This invention combines the compound of formula (I) or its precursor with glycyrrhizin or its derivatives for the first time, and found that it has a synergistic effect in enhancing the effect of RAR agonists. The inventors discovered for the first time that the transcriptional efficiency of RAR agonists in inflamed skin is structurally suppressed by NF-κB, and that when the composition is used in combination with RAR agonists, it can restore the transcriptional efficiency of RAR agonists through the RAR / RXR pathway, especially under chronic inflammatory conditions, thereby significantly enhancing the effect of RAR agonists.

[0026] 3. This invention combines the compound of formula (I) or its precursor with glycyrrhizin or its derivatives to form a composition and applies it to skin care, breaking through the technical limitations of using the two ingredients alone for skin care. The compound of formula (I) or its precursor and glycyrrhizin or its derivatives produce an unexpected synergistic effect, which improves the skin care effects such as skin homeostasis, anti-inflammatory effects, moisturizing, soothing and repairing, and whitening, far exceeding the efficacy of using the two ingredients alone.

[0027] 4. The composition provided by the present invention does not have independent effects on skin homeostasis, anti-inflammatory effects, moisturizing effects, soothing and repair effects, and whitening effects. Instead, they support and promote each other, forming a stable positive skin care cycle.

[0028] 5. The composition of the present invention can be used externally. The composition can be prepared into a variety of dosage forms such as aqueous solution, emulsion, ointment, cream, powder, oil, tablet, and lyophilized product. The dosage forms are rich and the application scenarios are wide, which can meet the usage needs of different groups of people in different scenarios. Attached Figure Description

[0029] Figure 1 The effects of urolithiasis, glycyrrhizin and their combined treatment on the expression levels of IL-1β, IL-6 and MMP1 genes in HSF cells; Figure 2 The effects of urolithiasis, glycyrrhizin and their combined treatment on the membrane potential of HaCaT cells; Figure 3 The effects of urolithiasis, glycyrrhizin and their combined treatment on melanin content and tyrosinase activity in B16 cells; Figure 4 The effects of urolithiasis, glycyrrhizin, retinol, and their combined treatments on the expression levels of IL-1β, IL-6, TNF-α, CDKN1A, KRT16, and MMP1 genes in HaCaT cells were investigated. Figure 5 The effects of combined treatment with urolithiasis, glycyrrhizin and retinol on the expression levels of CYP26A1 and CRABP2 genes in HaCaT cells were investigated. Figure 6The effect of combined treatment with urolithiasis, glycyrrhizin and adapalene on the expression level of CRABP2 gene in HaCaT cells; Figure 7 The effects of urolithiasis, glycyrrhizin, retinol, and their combined treatments on IL-6 expression levels in HSF cells were investigated. Detailed Implementation

[0030] Unless otherwise stated, the scientific and technical terms used in this disclosure shall have the meanings commonly understood by one of ordinary skill in the art. A particular term, without a specific definition, should not be considered uncertain or unclear, but should be understood in accordance with its common meaning in the art. When trade names appear herein, they are intended to refer to the corresponding product or its active ingredient.

[0031] definition Unless otherwise stated, the term "composition" as used herein may cover: products in which urolithin and glycyrrhizin are present in mixture form, and products in which urolithin and glycyrrhizin are packaged separately but may be mixed by the subject immediately prior to use or used together by the subject.

[0032] Unless otherwise specified, singular terms encompass plural terms, and plural terms encompass singular terms, such as the words “a,” “an,” or “the”, which refer to the plural referent.

[0033] The terms "optional" or "optionally" mean that an event or condition described subsequently may or may not occur, including both the occurrence and non-occurrence of the event or condition. For example, "optionally contains pharmaceutically or cosmetically acceptable excipients" means that the pharmaceutically or cosmetically acceptable excipients may or may not be contained.

[0034] The term "pharmaceutical acceptable" refers to a substance that is suitable for contact with the organs or tissues of a subject without causing excessive irritation, allergic reactions, immunogenicity, or toxicity, has a reasonable benefit / risk ratio, and will not adversely affect the intended use of the active ingredient (or active ingredient).

[0035] The term "cosmetically acceptable" refers to a substance that is suitable for contact with the skin or tissue of a subject without causing excessive irritation, allergic reactions, immunogenicity, or toxicity, and will not adversely affect the intended use of the active ingredient.

[0036] Unless otherwise stated, all figures used in the application documents to represent amounts of components, measurements, or reaction conditions should be understood to be modified by the term "about" in all cases, and the meaning of the term "about" here should be considered as within an acceptable margin of error for the corresponding value. When used with percentages, the term "about" may mean, for example, ±1%, preferably ±0.5%, more preferably ±0.1%.

[0037] As used herein, the term "subject" means any animal (e.g., a mammal) that is a recipient of a particular treatment, including but not limited to humans, non-human primates, canines, felines, rodents, etc. A subject can be a human.

[0038] Unless otherwise stated, the terms “comprise”, “comprises”, and “comprising” or their equivalents (contain, contain, containing, include, include, including) used herein are open-ended expressions, meaning that they may cover other unspecified elements, components, and steps in addition to those listed.

[0039] The present invention will now be described in detail through exemplary embodiments, but the scope of protection of the present invention is not limited thereto.

[0040] Combined use The compound of formula (I) described in this invention, or its salt, prodrug, metabolite or derivative thereof:

[0041] in: A, B, C, and D are each independently selected from H and OH; W, X, and Y are each independently selected from H and OH; and Z is selected from H and OH.

[0042] The inventors have discovered that the combination of the compound of formula (I) of the present invention with glycyrrhizin has a synergistic effect on improving stress and related outcomes, including significant synergistic improvement in cellular homeostasis / anti-inflammation, and further, significant synergistic improvement in mitochondrial homeostasis / anti-inflammation has also been observed.

[0043] It is understandable that stress can be cellular stress or oxidative stress. Stress-related outcomes can manifest as homeostasis / anti-inflammation, such as cellular homeostasis / anti-inflammation. Therefore, it can be observed that the combination of compound (I) and glycyrrhizin has a significant synergistic effect on mitochondrial homeostasis / anti-inflammation.

[0044] Based on this, on the one hand, the inventors further discovered that the combination of the compound of formula (I) and glycyrrhizin of the present invention exhibits a significant synergistic improvement effect on skin dryness, sensitivity, stinging, inflammation, and skin irritation. Specifically, the combination of the compound of formula (I) and glycyrrhizin can synergistically increase the water content of the stratum corneum and reduce transepidermal water loss, thereby achieving the effects of water retention and moisturizing; secondly, it can synergistically repair the skin barrier and inhibit inflammatory responses, thereby achieving the effects of maintaining skin homeostasis, anti-inflammation, and soothing repair. Furthermore, the repair of the skin barrier can further reduce water loss, making the moisturizing effect more lasting; at the same time, it can also synergistically and efficiently inhibit the production of melanin in the skin and promote melanin metabolism, thereby improving the whitening effect.

[0045] Therefore, the present invention provides a combined use: the use of a compound of formula (I) or a precursor thereof with glycyrrhizin or a derivative thereof in skin care.

[0046] In some preferred embodiments, the skin care includes maintaining skin homeostasis, anti-inflammatory effects, moisturizing, soothing and repairing effects, and skin whitening.

[0047] In some preferred embodiments, maintaining skin homeostasis includes maintaining the homeostasis of skin cells. More preferably, maintaining skin homeostasis includes maintaining the homeostasis of mitochondria.

[0048] On the other hand, the present invention also unexpectedly discovered that the combination of compound (I) and glycyrrhizin has a significant synergistic effect on the effect of RAR agonists.

[0049] Therefore, the present invention provides a combined use: the use of a compound of formula (I) or a precursor thereof with glycyrrhizin or a derivative thereof in enhancing the effect of RAR agonists.

[0050] The effects of the enhanced RAR agonist include promoting skin epidermal renewal or regeneration, improving wrinkles, improving skin elasticity and firmness, improving collagen loss or promoting collagen repair, improving or maintaining skin barrier function, improving biological aging, improving photoaging, improving uneven skin tone, improving acne, improving skin inflammation, and improving the skin matrix.

[0051] The effects of the enhanced RAR agonist include its effects on skin inflammation.

[0052] The skin inflammation is chronic skin inflammation or / and low-grade skin inflammation. For example, the skin inflammation may be caused by oxidative stress, ultraviolet radiation, aging, barrier damage, microecological imbalance, environmental pollution or aging; more preferably, the oxidative stress includes acute oxidative stress or chronic oxidative stress.

[0053] The effect of the enhanced RAR agonist is achieved by increasing or restoring transcriptional efficiency. Specifically, the effect of the enhanced RAR agonist is achieved by increasing or restoring the transcriptional efficiency of the RAR / RXR pathway.

[0054] The inventors discovered that when compound (I) is combined with glycyrrhizin and used in conjunction with a RAR agonist, compound (I) can reduce the activation level of NF-κB and decrease the entry of phosphorylated p65 into the cell nucleus to form a new inhibitory complex with RAR. Meanwhile, glycyrrhizin inhibits the phosphorylation and nuclear translocation of p65, reducing the formation of the complex. Therefore, when compound (I) is combined with glycyrrhizin as a PPARγ agonist, it activates the PPARγ / RXR pathway and indirectly inhibits NF-κB signal output.

[0055] Specifically, compound (I) exhibits multidimensional activity under stress conditions, simultaneously inhibiting the NF-κB / MAPK / ROS inflammatory signaling pathway and maintaining cellular chromatin homeostasis. By continuously inhibiting upstream activation flux of NF-κB and maintaining the chromatin microenvironment required for RAR transcription, it creates cellular preconditions for RAR transcriptional recovery. Glycyrrhizin, by inhibiting NF-κB p65 phosphorylation and nuclear translocation and activating the PPARγ / RXR pathway, relieves the interference of NF-κB on RAR / RXR transcriptional activity. Therefore, when compound (I) is combined with glycyrrhizin and used in conjunction with RAR agonists, the transcriptional efficiency of RAR agonists via the RAR / RXR pathway can be restored, especially the transcriptional efficiency of RAR agonists via the RAR / RXR pathway under chronic inflammatory conditions, thereby enhancing the effect of RAR agonists.

[0056] The aforementioned RAR agonists include RAR agonists or their pharmaceutically acceptable salts or derivatives.

[0057] In some preferred embodiments, the RAR agonist includes vitamin A-type RAR agonists and non-vitamin A-type RAR agonists.

[0058] RAR agonists can all bind to the retinoic acid receptor (RAR) and activate its transcriptional activity. The vitamin A-like RAR agonists are all chemically retinoids or their synthetic analogs, based on or mimicking the basic vitamin A skeleton, consisting of a β-ionone ring and conjugated polyene side chains. The non-vitamin A-like RAR agonists belong to the flavonoid class of compounds, differing from vitamin A-like structures, but they can mimic the functions of vitamin A-like compounds, exerting similar downstream effects as RAR agonists.

[0059] In some preferred embodiments, the vitamin A-type RAR agonists include retinoids (retinol, retinaldehyde, retinyl palmitate, retinyl linoleate, retinyl acetate, retinyl propionate, hydroxypinazone retinate (HPR), granactive retinoid (RR)), all-trans retinoic acid, adapalene, and trefarotin.

[0060] Retinol, molecular formula C 20 H 30 O is the alcohol form of vitamin A, which can be oxidized in the body in two steps to convert into retinal and all-trans retinoic acid, thereby exerting its biological effects. Retinal is an intermediate product of retinol oxidation and can be further oxidized to all-trans retinoic acid, making it an important link in the vitamin A-like metabolic transformation chain.

[0061] Retinyl palmitate is a retinyl ester derivative, retinyl linoleate is an ester derivative formed from retinol and linoleic acid, retinyl acetate is a retinyl ester that was used in industrial applications earlier, and retinyl propionate is an esterification product of retinol and propionate. All four are retinyl fatty acid esters, which are converted into retinoic acid in the body through retinol and retinal to exert their biological effects.

[0062] Hydroxypinazone retinyl ester (HPR) is a new generation of mild vitamin A ester that can directly bind to RAR receptors to take effect without stepwise conversion.

[0063] Retinol retinoic acid ester is a bimolecular retinoid formed by the esterification of one molecule of retinol and one molecule of retinoic acid. Part of it directly acts on RAR receptors, while the other part slowly decomposes to release retinol and trace amounts of retinoic acid.

[0064] All-trans retinoic acid (ATRA) is the final active metabolite of vitamin A in the body. It can directly bind to the retinoic acid receptor (RAR) and regulate the expression of target genes related to cell proliferation, differentiation, and apoptosis.

[0065] Adapalene, chemically known as 6-[3-(1-adamantyl)-4-methoxyphenyl]-2-naphthoic acid, has the molecular formula C2. 28 H 28 O3 is a naphthoic acid derivative that acts as a RARs agonist, specifically a selective RAR-β and RAR-γ agonist.

[0066] Trifarotene, chemically named 4-[3-(3-tert-butyl-4-pyrrolidinyl)phenyl-4-(2-hydroxyethoxy)]phenylbenzoic acid, is an agonist of the retinoic acid receptor (RAR) and has particularly selective activity for the γ-isomer of RAR.

[0067] In some preferred embodiments, the non-vitamin A RAR agonist includes iris isoflavones.

[0068] Tectorigenin (also known as iris aglycone) is a class of flavonoids derived from plants (such as Iris and kudzu). Chemically, tectorigenin belongs to the non-vitamin A class of compounds and can be used as an alternative or supplementary option for RAR agonists.

[0069] In some embodiments, in the combined use, the mass ratio of the compound of formula (I) or its precursor to glycyrrhizin or its derivative is (0.1-1000):1, preferably (1-100):1, more preferably (5-50):1.

[0070] In some embodiments, in the combined use, the mass ratio of the compound of formula (I) or its precursor to glycyrrhizin or its derivative is 0.1:1, 1:1, 5:1, 10:1, 50:1, 100:1, 200:1, 500:1, or 1000:1.

[0071] In some embodiments, the purity of the compound of formula (I) or its precursor is 10% or more, preferably 60% or more, more preferably 90% or more, for example 90% or more, 94% or more, 95% or more, 98% or more, or 100%.

[0072] In some embodiments, the purity of glycyrrhizin or its derivatives is 10% or more, preferably 60% or more, more preferably 90% or more, for example 90% or more, 94% or more, 95% or more, 98% or more, or 100%.

[0073] Compounds of formula (I) (urolithin) are metabolites produced by the gut microbiota of mammals (including humans) through the action of ellagitannins and ellagic acids. Ellagannins and ellagic acids are commonly found in foods such as pomegranates, nuts, and berries. Ellagannins themselves have extremely low absorption rates in the intestines. Urolithin is a class of compounds whose representative structural formula (I) is shown in the table below. Table 1 lists the structures of some common urolithins and provides explanations with reference to structural formula (I).

[0074] Table 1

[0075] Urolithin of any structure shown in structure (I) may be used in the combination or combination of the present invention.

[0076] In some combinations or arrangements of the present invention, suitable compounds are those of formula (I), wherein A, C, D and Z are independently selected from H and OH, respectively, and B, W, X and Y are all H, preferably, at least one of A, C, D and Z is OH.

[0077] A particularly suitable compound is naturally occurring urolithin. Therefore, Z is preferably OH, and W, X, and Y are preferably H. When W, X, and Y are all H, A and B are all H, and C, D, and Z are all OH, the compound is urolithin C. When W, X, and Y are all H, A, B, and C are all H, and D and Z are all OH, the compound is urolithin A. Preferably, the urolithin used in the method of this disclosure is urolithin A, urolithin B, urolithin C, or urolithin D. Most preferably, the urolithin used is urolithin A.

[0078] In this invention, the "precursor" of the compound of formula (I) refers to a substance whose molecular structure does not contain a urolithin core, but which can be biotransformed into urolithin by mammalian gut microbiota. In some embodiments, the precursors of urolithin include ellagic acid, ellagitannins, and combinations thereof. Ellagic acid and ellagitannins are found in fruits such as pomegranates, raspberries, cloudberries, and blackberries, as well as nuts such as walnuts and pistachios. Urolithin cannot be directly extracted from fruits such as pomegranates, raspberries, cloudberries, blackberries, walnuts, and pistachios, but its precursors, ellagic acid and ellagitannins, can be directly extracted from fruits such as pomegranates, raspberries, cloudberries, blackberries, walnuts, and pistachios. Furthermore, ellagic acid and ellagitannins can be converted into urolithin in vivo, thereby exerting the corresponding effects.

[0079] Currently, urolithin is mainly obtained through artificial synthesis. The mainstream method is chemical synthesis, which has the advantages of high purity (over 99.5%), no limitation by intestinal flora, large yield, controllable cost, and good stability. Alternatively, it can be synthesized using bio-fermentation / enzyme catalysis, where ellagic acid / ellagitannins are used as raw materials, and specific engineered bacteria / intestinal bacteria are used for enzymatic hydrolysis and fermentation to convert them into urolithin. This method has low conversion rate, long cycle time, and is difficult to scale up, making it unsuitable for large-scale production. In some embodiments, at least a portion of the urolithin is obtained through chemical synthesis. Furthermore, or optionally, at least a portion of the urolithin can be obtained through bio-fermentation / enzyme catalysis.

[0080] Besides urolithin, compounds of formula (I) may also include salts, prodrugs, metabolites, or derivatives of urolithin, which have similar structures to urolithin and all possess the same polyphenol core structure. Among them, urolithin salts are ionic compounds formed by the acid-base neutralization of free urolithin with alkali metals, organic bases, etc. Typical examples include sodium salt, potassium salt, choline salt, lysine salt, and magnesium salt of urolithin A, which improve water solubility and enhance dissolution and absorption performance compared to free urolithin.

[0081] Urolithin prodrugs are obtained by covalently modifying the phenolic hydroxyl groups on the urolithin nucleus. They have very low intrinsic activity. Once in the body, they are hydrolyzed / metabolic by esterases and oxidases, removing the modified groups and releasing the active urolithin. Compared to free urolithin, urolithin prodrugs improve water solubility, metabolic stability, intestinal absorption, avoid first-pass metabolism, and prolong the duration of action. Typical examples include urolithin A diacetate, urolithin A phosphate prodrugs, and urolithin A glucoside prodrugs.

[0082] Urolithin metabolites are natural in vivo products generated from free urolithin through biotransformation in the liver and intestines after it enters the human body. The two main metabolic pathways are glucuronidation and sulfation. Typical examples include urolithin A-3-O-glucuronide, urolithin A-8-O-glucuronide, urolithin A sulfate, urolithin B glucuronic acid, and urolithin B sulfate.

[0083] Urolithin derivatives are compounds obtained by covalently modifying the urolithin core, and cannot be reverted to the original urolithin after the modification is removed. Typical examples include 3-methoxyurolithin A and brominated / fluorinated urolithin A.

[0084] In the above-mentioned combined applications, urolithin, urolithin precursors, or various combinations of urolithin and urolithin precursors can be used. However, the dosage calculation is uniformly based on the amount of urolithin.

[0085] Glycyrrhizin is a flavonoid compound extracted from the root of *Glycyrrhiza glabra*. It inhibits tyrosinase activity to reduce melanin production and possesses both antioxidant and anti-inflammatory properties, making it suitable as a natural antioxidant. Glycyrrhizin and its derivatives can be derived from any suitable source and can be isolated or chemically synthesized. In some embodiments, the glycyrrhizin and its derivatives are isolated and purified from the root of *Glycyrrhiza glabra*. In other embodiments, the glycyrrhizin and its derivatives can also be obtained through artificial chemical synthesis or semi-synthesis. Non-limiting examples of suitable glycyrrhizin derivatives include 5'-formyl glycyrrhizin, glycyrrhizin dimers, 5'-isoprendyl glycyrrhizin, and R-glycyrrhizin, and mixtures thereof.

[0086] In the aforementioned combined uses, glycyrrhizin alone, glycyrrhizin derivatives alone, or various combinations of glycyrrhizin and its derivatives can be used. However, the dosage is uniformly calculated based on the amount of glycyrrhizin.

[0087] Composition Based on the synergistic effect of the above-mentioned compound of formula (I) or its precursor and glycyrrhizin or its derivative, the present invention provides a composition comprising: compound of formula (I) or its precursor, and glycyrrhizin or its derivative.

[0088] Based on the synergistic effect of the above-mentioned compound of formula (I) or its precursor and glycyrrhizin or its derivative in enhancing the effect of RAR agonists, the present invention provides a composition comprising: compound of formula (I) or its precursor, glycyrrhizin or its derivative, and RAR agonist.

[0089] In some embodiments, the mass ratio of the compound of formula (I) or its precursor to glycyrrhizin or its derivative is (0.1-1000):1, preferably (1-100):1, and more preferably (5-50):1.

[0090] In some embodiments, in the combined use, the mass ratio of the compound of formula (I) or its precursor to glycyrrhizin or its derivative is 0.1:1, 1:1, 5:1, 10:1, 50:1, 100:1, 200:1, 500:1, or 1000:1.

[0091] In some embodiments, the mass percentage of the compound of formula (I) or its precursor in the composition is 0.01%-5%, preferably 0.01% to 1%, more preferably 0.1% to 1%, for example 0.01%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%.

[0092] In some embodiments, the mass percentage of glycyrrhizin or its derivatives in the composition is 0.001% to 1%, preferably 0.005% to 0.5%, more preferably 0.01% to 0.1%, for example 0.001%, 0.01%, 0.05%, 0.1%, 0.5%, 1%.

[0093] In some embodiments, the composition comprises, by mass percentage, a compound of formula (I) or a precursor thereof and, by mass percentage, glycyrrhizin or a derivative thereof. For example, the composition comprises, by mass percentage, 0.5% urolithin A and 0.1% glycyrrhizin, or both of these components as active ingredients.

[0094] In some embodiments, the purity of the compound of formula (I) or its precursor is 10% or more, preferably 60% or more, more preferably 90% or more, for example 90% or more, 94% or more, 95% or more, 98% or more, or 100%.

[0095] In some embodiments, the purity of glycyrrhizin or its derivatives is 10% or more, preferably 60% or more, more preferably 90% or more, for example 90% or more, 94% or more, 95% or more, 98% or more, or 100%.

[0096] In some embodiments, the compound of formula (I) or its precursor, such as urolithin and its precursors, can be derived from any suitable source, and can be isolated and / or chemically synthesized, or derived from in vivo conversion. In some embodiments, the precursor of urolithin is obtained directly from fruits such as pomegranate, raspberry, cloudberry, blackberry, walnut, pistachio, etc. In some embodiments, at least a portion of the urolithin is obtained by chemical synthesis. Additionally or alternatively, at least a portion of the urolithin can be obtained by biofermentation / enzymatic catalysis.

[0097] In some embodiments, glycyrrhizin and its derivatives can be derived from any suitable source, and can be isolated or chemically synthesized. In some embodiments, the glycyrrhizin and its derivatives are isolated and purified from the root of *Glycyrrhiza glabra*. In other embodiments, the glycyrrhizin and its derivatives can also be obtained by artificial chemical synthesis or semi-synthesis. Non-limiting examples of suitable glycyrrhizin derivatives include 5'-formylglycyrrhizin, glycyrrhizin dimers, 5'-isoprendylglycyrrhizin, and R-glycyrrhizin and mixtures thereof.

[0098] In another embodiment, a composition containing a RAR agonist is applied topically, wherein the RAR agonist content is 0.005% (w / w) to 0.5% (w / w) of the composition, for example 0.01% (w / w) to 0.1% (w / w), 0.02% (w / w) to 0.08% (w / w), or 0.03% (w / w) to 0.07% (w / w), or 0.04% (w / w) to 0.06% (w / w), for example 0.01% (w / w), 0.03% (w / w), 0.05% (w / w), 0.07% (w / w), or 0.1% (w / w).

[0099] Product / Formulation In some embodiments, the present invention provides a product or formulation comprising the composition described herein.

[0100] In some embodiments, the present invention provides a homeostatic / anti-inflammatory product or formulation comprising the composition described herein. In some embodiments, the homeostatic / anti-inflammatory effect includes maintaining cellular homeostasis / anti-inflammatory activity. In some embodiments, the homeostatic / anti-inflammatory effect includes maintaining mitochondrial homeostasis / anti-inflammatory activity.

[0101] In some embodiments, the present invention provides a skin care product or formulation comprising the compositions described herein. In some embodiments, the skin care includes maintaining skin homeostasis, reducing skin inflammation, moisturizing the skin, soothing and repairing the skin, and whitening the skin.

[0102] In some embodiments, the product or formulation is a liquid, solid, or semi-solid product or formulation.

[0103] In some embodiments, the product or formulation is a topical product or formulation.

[0104] Those skilled in the art will understand that, depending on the type of product or formulation, the product or formulation may also include various types of acceptable excipients. Acceptable excipients may include, for example, solvents, diluents, antibacterial agents, excipients, buffers, fillers, binders, disintegrants, preservatives, lubricants, odorants, flavorings, etc.

[0105] In some embodiments, the topical product or preparation is a cosmetic.

[0106] In some embodiments, in the product or formulation, particularly in a topical product or formulation, such as a cosmetic, the mass content of the compound of formula (I) or its precursor is 0.01%-5% relative to the total mass of the product or formulation, preferably 0.01%-1%, more preferably 0.1%-1%, for example, 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%.

[0107] In some embodiments, in the product or formulation, particularly in a topical product or formulation, such as a cosmetic, the mass content of glycyrrhizin or its derivative is 0.001%-1%, preferably 0.005%-0.5%, more preferably 0.01%-0.1%, relative to the total mass of the product or formulation. For example, it can be 0.001%, 0.005%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.5%, or 1%.

[0108] When the products or formulations of this invention are provided as cosmetics, they can be used as aqueous solutions, lotions, sprays, or suspensions. Cosmetics can be prepared in various desired forms, such as liquids, powders, granules, and lumps; semi-solids such as creams and pastes; and gels. Such cosmetics include basic cosmetics such as facial cleansers, lotions, face creams, eye creams, neck creams, hand creams, gels, moisturizers, serums / essences (beauty serums), sprays, masks, and freeze-dried essences; color cosmetics such as foundations, toners, facial oils, and lipsticks; oral cosmetics (mouthwash or toothpaste); fragrance cosmetics; hair cosmetics (shampoos, conditioners, scalp treatments); and body cosmetics (shower gels, body lotions). Cosmetics containing urolithin and glycyrrhizin according to this invention can be used as moisturizing cosmetics, whitening cosmetics, and repairing cosmetics.

[0109] In some embodiments, the cosmetic is a serum for skin care. The serum contains, by weight percentage, 0.05-1% urolithin, 0.001-0.1% glycyrrhizin or a derivative thereof, and cosmetically acceptable excipients. For example, a serum, by weight percentage, comprises the following components: 0.05-1% urolithin, 0.001-0.1% glycyrrhizin or a derivative thereof, 0.05%-5% thickening / suspending agent, 2%-20% moisturizer, 0.1%-3% preservative, and the balance being water.

[0110] In some embodiments, the cosmetic is a face cream for skin care. The face cream contains, by weight percentage, 0.05-1% urolithin, 0.001-0.1% glycyrrhizin or its derivatives, and cosmetically acceptable excipients. For example, in some embodiments, the face cream contains, by weight percentage, 0.05-1% urolithin, 0.001-0.1% glycyrrhizin or its derivatives, 1%-15% moisturizing system, 1%-10% emulsifying oil system, 1%-8% emulsifying and thickening system, 0.1%-3% polyol solubilizer / preservative, 0.01%-0.1% chelating agent, and the balance being water.

[0111] In some embodiments, the cosmetic is a cream. The cream contains, by weight percentage, 0.05-1% urolithiasis, 0.001-0.1% glycyrrhizin or its derivatives, and an aqueous phase, an oil phase, and an emulsifier. The aqueous phase includes, but is not limited to, water, polyols, disodium EDTA, and ammonium acryloylmethyl taurate / VP copolymer. The oil phase includes, but is not limited to, one or more of caprylic / capric triglycerides, isononyl isononanoate, hydrogenated polydecene, polydimethylsiloxane, and cyclopentamethoxysiloxane. The emulsifier includes, but is not limited to, cetearyl alcohol, cetearyl glucoside, cetearyl oleate, and sorbitan oleate. Preferably, the polyol includes, or is not limited to, glycerol, butylene glycol, 1,2-pentanediol, and 1,2-hexanediol.

[0112] In some embodiments, the cream comprises, by weight percentage: 0.05-1% urolithiasis, 0.001-0.1% glycyrrhizin or its derivatives, 2-5% glycerin, 2-5% butylene glycol, 1-3% cetearyl alcohol, 1-3% isononyl isononate, 1-3% hydrogenated polydecene, 1-3% polydimethylsiloxane, 1-3% cyclopentamethoxydimethylsiloxane, 0.1-0.5% 1,2-pentanediol, 0.1-0.5% 1,2-hexanediol, 0.5-3% cetearyl glucoside, 0.1-0.5% ammonium acryloylmethyl taurate / VP copolymer, 0.01-0.05% disodium EDTA, with the balance being water.

[0113] The compositions, products, or formulations of the present invention can be packaged in suitable containers, such as bottles, bags, cans, spray cans, spray containers, boxes, bags, etc., as needed.

[0114] In some embodiments, when the product or formulation is used as a cosmetic, it optionally includes cosmetically acceptable excipients. In some embodiments, the cosmetically acceptable excipients include, but are not limited to, moisturizers, emollients, whitening agents, anti-wrinkle agents, antioxidants, sunscreens, free radical scavengers, preservatives, surfactants, fragrances, pigments, or thickeners, or other ingredients used in cosmetics.

[0115] Formulation combination The present invention also provides a formulation comprising: (1) A first formulation containing a compound of formula (I) or a precursor thereof; and (2) A second preparation containing glycyrrhizin or its derivatives.

[0116] Furthermore, the above-mentioned formulation combination also includes: (3) instructions, which instruct the subject to administer the first formulation and the second formulation.

[0117] The present invention also provides a formulation combination comprising: (1) A first formulation containing a compound of formula (I) or a precursor thereof; (2) A second preparation containing glycyrrhizin or its derivatives; and (3) A third formulation containing a RAR agonist.

[0118] In this invention, the aforementioned objects may include objects that need to address skin problems, anti-aging, anti-inflammation, moisturizing, whitening, anti-oxidative stress, and / or maintain skin homeostasis.

[0119] In this invention, the objects may include those that require anti-skin aging, skin barrier repair, skin moisturizing, skin whitening, anti-skin inflammation, or skin anti-oxidation, or those that require improvement of skin aging caused by inflammation or skin aging caused by oxidative stress.

[0120] In this invention, the above-mentioned formulation combination can be packaged separately and sold separately for the first, second and third formulations; or the first, second and third formulations can be prepared separately but packaged into one outer package and sold as a combination; or the first, second and third formulations can be mixed to prepare a composition or a product or formulation in the manner described above as a composition or product or formulation.

[0121] In this invention, the specific contents of the specification may include the recommended dose ratio of the compound of formula (I) or its precursor to glycyrrhizin or its derivative.

[0122] In this invention, the recommended dosage ratio can be referenced to the mass ratio of the components in the above composition.

[0123] In this invention, the specific contents of the specification may also include the target of application, the method of application, the dosage of application, the timing and frequency of application, etc.

[0124] How to use The present invention also provides a method for addressing skin problems, or for anti-inflammatory, anti-oxidative stress, moisturizing, whitening, and maintaining skin homeostasis, comprising: applying an effective amount of the composition, product or formulation described herein, or combination of the formulations described herein to a subject in need.

[0125] In some embodiments, the present invention provides a skin care method comprising applying to the skin of a subject in need a compound of formula (I) or a precursor thereof and glycyrrhizin or a derivative thereof, the composition, the product or / and formulation, or a combination of the formulations.

[0126] In the above embodiments, the skin care method can achieve the following: anti-skin aging, skin barrier repair, skin barrier improvement, moisturizing, whitening, anti-skin inflammation, skin anti-oxidation, and maintaining skin homeostasis.

[0127] In some preferred embodiments of the present invention, the composition, the product or formulation, or the compound of formula (I) or its precursor and glycyrrhizin or its derivative in the formulation combination are administered by any of the following methods: external application.

[0128] In some other preferred embodiments of the present invention, the external application method is to apply it by smearing.

[0129] In some preferred embodiments of the present invention, the compound of formula (I) or its precursor and glycyrrhizin or its derivatives may be applied in the same manner or in different manners.

[0130] Those skilled in the art will understand that the compound of formula (I) or its precursor and glycyrrhizin or its derivatives may be administered simultaneously or in different orders (i.e., at regular or irregular intervals). The term "order" means that the compound of formula (I) or its precursor and glycyrrhizin or its derivatives are administered in a continuous manner. The time interval between two consecutive administrations may be, for example, a second or several seconds, minutes or hours on the same day; a day or several days or weeks in the same month; or a month or several months in the same year.

[0131] In another embodiment, the composition of the present invention is administered topically (e.g., on the skin of a subject).

[0132] Those skilled in the art will understand that the subjects who may be required to participate include mammals, preferably humans or pets.

[0133] The term "pet" means any animal that can benefit from or enjoy the compositions provided in this disclosure. For example, a pet can be a bird, a cow, a dog, a horse, a cat, an antelope, a wolf, a rodent, a sheep, or a pig, but a pet can be any suitable animal.

[0134] In one implementation, the human being is female, such as an adolescent female, a middle-aged female, a menopausal or perimenopausal female, or an elderly female.

[0135] In the method of the present invention, the human being may also include, for example, a person with photoaging or radiation-induced skin aging, or a person suffering from skin inflammation, or a person suffering from skin damage, or a person suffering from oxidative stress.

[0136] use Mitochondria are core organelles for maintaining cellular homeostasis. Under persistent oxidative stress, mitochondrial function becomes abnormal, leading to homeostasis imbalance. Furthermore, excessive free radicals continuously damage mitochondrial DNA and biological membranes, further exacerbating mitochondrial structural damage. Simultaneously, they release mitochondrial damage-related molecules, activate inflammasomes, and promote the massive release of inflammatory factors such as TNF-α and IL-6.

[0137] The compounds of formula (I) obtained by means of this invention and glycyrrhizin, compositions containing them, the products or formulations thereof, or combinations of the formulations thereof may be used to improve stress (e.g., cellular stress) and related outcomes.

[0138] Urolithin or its precursors and glycyrrhizin or its derivatives obtained by the present invention, compositions containing them, said products or formulations, or combinations of said formulations, can be used to improve oxidative stress, such as acute oxidative stress or chronic oxidative stress and related outcomes.

[0139] The compounds of formula (I) obtained by the present invention or their precursors and glycyrrhizin or its derivatives, compositions containing them, the products or formulations thereof, or combinations of the formulations thereof may be used for steady-state / anti-inflammatory purposes.

[0140] The compounds of formula (I) obtained by the present invention or their precursors and glycyrrhizin or its derivatives, compositions containing them, the products or formulations thereof, or combinations of the formulations thereof may be used for cell homeostasis / anti-inflammatory purposes.

[0141] The compounds of formula (I) obtained by the present invention or their precursors and glycyrrhizin or its derivatives, compositions containing them, the products or formulations thereof, or combinations of the formulations thereof may be used for mitochondrial homeostasis / anti-inflammatory purposes.

[0142] The compounds of formula (I) obtained by the present invention or their precursors and glycyrrhizin or its derivatives, compositions containing them, the products or formulations thereof, or combinations of the formulations thereof may be used for skin care purposes.

[0143] The compound of formula (I) obtained by the present invention or its precursor and glycyrrhizin or its derivatives, compositions containing them, the products or preparations thereof, or combinations of the preparations thereof may be used for skin homeostasis, skin anti-inflammation, skin moisturization, skin soothing and repair, and skin whitening purposes.

[0144] In some embodiments, the present invention provides the use of a compound of formula (I) or a precursor thereof and glycyrrhizin or a derivative thereof, the composition, the product or formulation, or the combination of the formulations in the preparation of an article for homeostasis / anti-inflammatory purposes.

[0145] In some embodiments, the present invention provides the use of a compound of formula (I) or a precursor thereof and glycyrrhizin or a derivative thereof, the composition, the product or formulation, or the combination of the formulations in the preparation of an article for cell homeostasis / anti-inflammatory purposes.

[0146] In some embodiments, the present invention provides the use of a compound of formula (I) or a precursor thereof and glycyrrhizin or a derivative thereof, the composition, the product or formulation, or the combination of the formulations in the preparation of an article for mitochondrial homeostasis / anti-inflammatory purposes.

[0147] In some embodiments, the present invention provides the use of a compound of formula (I) or a precursor thereof and glycyrrhizin or a derivative thereof, the composition, the product or formulation, or the combination of the formulations in the preparation of an article for skin care.

[0148] In some embodiments, the present invention provides the use of a compound of formula (I) or a precursor thereof and glycyrrhizin or a derivative thereof, the composition, the product or formulation, or the combination of the formulations in the preparation of articles for skin homeostasis, skin anti-inflammation, skin moisturizing, skin soothing and repair, and skin whitening.

[0149] In some embodiments, the present invention provides the use of a compound of formula (I) or a precursor thereof and glycyrrhizin or a derivative thereof, the composition, the product or formulation, or the combination of the formulation in the preparation of articles for promoting skin epidermal renewal or regeneration, improving wrinkles, improving skin elasticity and firmness, improving collagen loss or promoting collagen repair, improving or maintaining skin barrier function, improving biological aging, improving photoaging, improving uneven skin tone, improving acne, improving skin inflammation, and improving the skin matrix.

[0150] In some embodiments, the present invention also provides a non-therapeutic skin care method comprising applying the composition of the present invention. The present invention also provides a method for: promoting skin epidermal renewal or regeneration, improving wrinkles, improving skin elasticity and firmness, improving collagen loss or promoting collagen repair, improving or maintaining skin barrier function, improving biological aging, improving photoaging, improving uneven skin tone, improving acne, improving skin inflammation, and improving the skin matrix, comprising applying the composition of the present invention.

[0151] In some embodiments, the present invention also provides a non-therapeutic method for enhancing the effect of a RAR agonist, the method comprising administering the composition of the present invention. The present invention also provides a method for enhancing the effect of a RAR agonist, comprising administering the composition of the present invention.

[0152] RAR agonists play an important role in regulating a variety of biological processes, including cell proliferation, differentiation, apoptosis, inflammation, and development.

[0153] RAR agonists are commonly used in cosmetic formulations to reduce wrinkles and improve cellulite, and have been approved by the U.S. Food and Drug Administration (FDA) for anti-aging treatments since 1996. The anti-aging and anti-photoaging effects of RAR agonists are based on their ability to promote keratinocyte proliferation, collagen and elastin fiber synthesis, and inhibit collagen degradation by suppressing metalloproteinase activity. These effects ultimately slow down the aging process, enhance repair mechanisms, and reduce wrinkles and pigmentation.

[0154] The effects of the enhanced RAR agonist include promoting skin epidermal renewal or regeneration, improving wrinkles, improving skin elasticity and firmness, improving collagen loss or promoting collagen repair, improving or maintaining skin barrier function, improving biological aging, improving photoaging, improving uneven skin tone, improving acne, improving skin inflammation, and improving the skin matrix.

[0155] Example 1: Topical skin care cream Table 2

[0156] Preparation method The oil phase raw materials (cetearearyl alcohol, cetearyl glucoside, isononyl isononanoate, hydrogenated polydecene, polydimethylsiloxane, cyclopentamethoxysiloxane) are heated to 80°C and stirred until completely dissolved and transparent. The aqueous phase raw materials (deionized water, glycerin, 1,3-butanediol, disodium EDTA, ammonium acryloylmethyl taurate / VP copolymer, urolithin A) are heated to 80°C and stirred until completely dissolved. The aqueous phase is slowly added to the oil phase and homogenized and emulsified until fine. The mixture is then cooled to 45°C and pre-dissolved glycyrrhizin, 1,2-pentanediol, and 1,2-hexanediol are added. After stirring evenly, the mixture is cooled and discharged to obtain the skin care cream.

[0157] Comparative Example 1: Topical White Cream Table 3

[0158] The preparation method is the same as in Example 1.

[0159] Example 2: Topical Skin Care Essence Table 4

[0160] Preparation method Urolithiasis A was dispersed evenly in glycerol, 1,3-butanediol, and deionized water. Then, panthenol, sodium hyaluronate, disodium EDTA, xanthan gum, and hydroxyethyl cellulose were added. The mixture was heated to 80°C and stirred until completely dissolved. When the system temperature dropped below 45°C, 1,2-pentanediol, 1,2-hexanediol, and 1,3-butanediol pre-dissolved glycyrrhizin were added. The mixture was stirred until homogenized, then cooled to room temperature and filtered to obtain the skin care essence.

[0161] Example 3: Topical Skin Care Cream Table 5

[0162] Preparation method Deionized water, glycerin, 1,3-butanediol, panthenol, sodium hyaluronate, and disodium EDTA are heated to 80°C and stirred until completely dissolved to obtain an aqueous phase. Cetearyl alcohol, squalane, phytosterol oleate, polydimethylsiloxane, and glyceryl stearate are heated to 80°C and stirred until completely dissolved to obtain an oil phase. The oil phase is slowly added to the aqueous phase and homogenized and emulsified until smooth. The mixture is then cooled to 35°C and pre-dissolved urolithin A, glycyrrhizin, 1,2-pentanediol, and 1,2-hexanediol are added. After stirring evenly and cooling, the mixture is discharged to obtain a skin care cream.

[0163] Example 4: A Steady-State / Anti-inflammatory Method This embodiment provides a steady-state / anti-inflammatory method, including the topical application of the skin care cream described in Example 1.

[0164] Example 5: An Essence Table 6

[0165] Preparation method: 1. A portion of glycerol, polysorbate-80, urolithin A, and polyvinylpyrrolidone were dispersed using a high-speed homogenizer; 2. Mix the remaining glycerin, polysorbate-80, glycyrrhizin, and tocopheryl acetate for later use; 3. Mix and disperse 1,3-butanediol and xanthan gum, add deionized water and p-hydroxyacetophenone, as well as the mixture from step 1, heat to 90°C, keep warm for 20 min for sterilization, homogenize at 5000 rpm for 2 min, and stir to cool. 4. Cool down to 45℃, add the mixture from step 2 and 1,2-hexanediol in sequence, stir evenly and then discharge.

[0166] In addition, an extract containing 0.05% (w / w) retinaldehyde can be provided, which can be prepared by mixing it with glycyrrhizin.

[0167] Example 6: An emulsion Table 7

[0168] Preparation method: 1. Disperse a portion of glycerol, a portion of deionized water, and urolithin A using a high-speed homogenizer; 2. Mix and dissolve 1,3-butanediol, polysorbate-80, and glycyrrhizin for later use; 3. Prepare the aqueous phase: Mix and disperse xanthan gum and remaining glycerol, then add the remaining deionized water and p-hydroxyacetophenone, along with the mixture from step 1. Heat to 90°C and sterilize for 20 minutes. Homogenize at 5000 rpm for 2 minutes, and keep warm for later use. 4. Add cetearyl oleate, sorbitan oleate, caprylic / capric triglyceride, isononyl isononanoate, and cyclopentamethoxysiloxane to another beaker and heat until the solids dissolve. Add to the aqueous phase, homogenize at 5000 rpm for 2 min, and stir to cool.

[0169] 5. Cool down to 40℃, add the mixture from step 2 and 1,2-hexanediol in sequence, stir evenly and then discharge.

[0170] In addition, a cream containing 0.3% (w / w) retinyl palmitate can be provided, which can be prepared by mixing it with glycyrrhizin.

[0171] Example 7: A cream Table 8

[0172] Preparation method: 1. A portion of glycerol, polysorbate-80, urolithin A, and polyvinylpyrrolidone were dispersed using a high-speed homogenizer; 2. Mix 1,3-butanediol and glycyrrhizin for later use; 3. Preparation of aqueous phase: After swelling the ammonium acryloyl dimethyl taurate / VP copolymer in cold water for 15 min, heat it to 90℃ and keep it at that temperature for 20 min to sterilize it. Homogenize it at 5000 rpm for 1 min, add p-hydroxyacetophenone and the mixture from step 1, homogenize it at 5000 rpm for 2 min, and keep it at that temperature for later use. 4. Add cetearyl alcohol, cetearyl glucoside, glyceryl stearate, PEG-100 stearate, caprylic / capric triglyceride, isononyl isononanoate, and cyclopentamethoxysiloxane to another beaker and heat until the solids dissolve. Add the solids to the aqueous phase, homogenize at 5000 rpm for 2 min, and stir to cool. 5. Cool down to 40℃, add the mixture from step 2 and 1,2-hexanediol in sequence, stir evenly and then discharge.

[0173] In addition, a cream containing 0.3% (w / w) retinyl palmitate can be provided, which can be prepared by mixing it with glycyrrhizin.

[0174] Example 8: An oil cream Table 9

[0175] Preparation method: 1. Prepare the oil phase: Add cyclopentamethoxydimethylsiloxane, dioctyl ether, isononyl isononyl ether, polyglycerol-3 diisostearate, lauryl PEG-10 tris(trimethylsiloxy)silylethyl polydimethylsiloxane and octanoic acid / capric triglyceride to a beaker and stir until homogeneous; 2. Disperse a portion of glycerol, polysorbate-80, urolithiasis A, and a portion of deionized water evenly. 3. Mix 1,3-butanediol and glycyrrhizin for later use; 4. Prepare the aqueous phase: Mix the remaining glycerol, ammonium acryloyl dimethyl taurate / VP copolymer, and p-hydroxyacetophenone evenly, add the remaining water and the mixture from step 2, heat to 90°C, and stir until homogenized. 5. Slowly pour the aqueous phase into the oil phase while stirring, and homogenize at 5000 rpm for 2 minutes; 6. Cool down to below 45°C, add 1,2-hexanediol and the mixture from step 3, stir evenly, and then cool to room temperature before discharging.

[0176] In addition, a cream containing 1.0% (w / w) hydroxypinazone retinate can be provided, which can be prepared by stirring it evenly with cyclopentamethoxysiloxane or the like.

[0177] The concentration conversion relationships of urolithiasis AC, glycyrrhizin, retinol, GW9662, and BAY 11-7082 in the following experimental examples are shown in Table 10.

[0178] Table 10

[0179] Experimental Example 1: Synergistic effect of urolithiasis and glycyrrhizin on anti-aging and anti-inflammatory effects in skin cells. Aging Model Establishment and Preprocessing

[0180] HSF cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum. HSF cells were seeded into 96-well plates with D-gal concentrations of 0, 50, 100, 200, 300, 400, and 500 mM, respectively. After 48 h of treatment, the medium was changed and 10% CCK-8 reagent was added. OD was measured 1 h later. 450 And calculate cell viability and IC50. 30The experiment was set up with 3 replicates, and the results showed that the IC50 of D-gal was... 30 The initial concentration was 152.15 mM, and 150 mM was selected as the model induction concentration for subsequent experiments.

[0181] Combined drug treatment HSF cells in the logarithmic growth phase were harvested, and after reaching 50% confluence, the medium was changed and the cells were treated as follows: (1) NC group: No special treatment; (2) D-gal group: Add 150 mM D-gal; (3) UA group: 150 mM D-gal and 2 μM urolithiasis A were added simultaneously; (4) UB group: 150 mM D-gal and 2 μM urolithin B were added simultaneously; (5) GLD group: 150 mM D-gal and 5 μM glycyrrhizin were added simultaneously; (6) GLD+UA group: 150 mM D-gal, 2 μM urolithin A and 5 μM glycyrrhizin were added simultaneously; (7) GLD+UB group: 150 mM D-gal, 2 μM urolithin B and 5 μM glycyrrhizin were added simultaneously.

[0182] Cells were treated at 37℃ and 5% CO2 for 48 h, and total RNA was extracted from each group. The expression levels of IL-1β, IL-6, and MMP1 were detected by qPCR (β-actin was used as the internal control). The experiment was performed in ≥3 replicates to evaluate the synergistic effect of urolithiasis and glycyrrhizin combined treatment on anti-aging and anti-inflammatory effects. The results were analyzed using one-way ANOVA to test the significance of differences between groups (P < 0.05 was considered statistically significant). Bliss analysis of independence, a commonly used statistical model in biology, was used to further confirm the synergistic effect of the combination.

[0183] Experimental results The results are as follows Figure 1 As shown, D-gal treatment significantly upregulated IL-1β, IL-6, and MMP1 (P<0.001), indicating abnormal cell function and successfully inducing senescence. Urolithin A, urolithin B, and glycyrrhizin had some inhibitory effect on IL-1β and IL-6 gene expression in the D-gal-induced HSF cell senescence model, but the effect was not significant. However, they significantly inhibited MMP1 gene expression (P<0.0001). The inhibitory effect was more significant when urolithin A and glycyrrhizin were used in combination, or when urolithin B and glycyrrhizin were used in combination, compared to the inhibitory effect of using them alone.

[0184] Furthermore, the protective rates and effects of the combination of urolithin A and glycyrrhizin and the combination of urolithin B and glycyrrhizin were calculated to further determine the synergistic effect of the combination.

[0185] Core assumption: Within the 10-20 μM range, the drug regulation rate is linearly positively correlated with concentration (the standard simplified assumption when no concentration gradient data is available); RE: Represents Relative Expression, i.e., the expression level of the target gene (such as IL-1β, IL6, MMP1) relative to the internal reference gene (β-actin), calculated by qPCR (2^-ΔΔCt method). Quantification of the experimental group's damage repair efficiency: Using commonly used biological statistical formulas, synergistic effects can be determined: E Bliss =E A +E B - (E A ×E B )

[0186] Among them, E A E B The protection rate of two single drugs, E Bliss This refers to the protection rate when two drugs are theoretically combined for synergistic treatment.

[0187] If the combined processing protection rate > E Bliss This indicates a trend of synergistic effect.

[0188] The synergistic results are shown in Table 11. It can be seen that the combined protection rates of urolithin A or urolithin B with glycyrrhizin against IL-1β, IL6, and MMP1 are all greater than the theoretical protection rates, indicating that they all have a synergistic effect.

[0189] Table 11 Bliss Independence Analysis of IL-1β, IL-6, and MMP1

[0190] The above experiments revealed that D-gal treatment significantly upregulated the expression levels of IL-1β, IL-6, and MMP1. IL-1β and IL-6 are classic pro-inflammatory cytokines, and their upregulation is an important marker of chronic low-grade inflammation (one of the core characteristics of skin aging). The significant upregulation of IL-1β and IL-6 expression caused by D-gal treatment further confirms that D-gal not only directly induces cell cycle arrest but also exacerbates the aging process by activating inflammatory pathways. MMP1 is a marker of extracellular matrix remodeling. Furthermore, MMP1 is a key protease responsible for collagen breakdown in the human body. The significant upregulation of MMP1 expression indicates that chronic inflammation-induced tissue damage can also induce skin aging and wrinkles, making it a core biomarker of skin aging. The significant upregulation of IL-1β, IL-6, and MMP1 caused by D-gal treatment confirms that D-gal treatment successfully constructed a cellular aging model.

[0191] In a HSF cell senescence model, treatment with a combination of urolithin A and glycyrrhizin significantly downregulated the expression levels of IL-1β, IL-6, and MMP1. Bliss independence analysis showed that the combined use of urolithin and glycyrrhizin had a synergistic effect in inhibiting the expression levels of IL-1β, IL-6, and MMP1.

[0192] Experimental Example 2: Synergistic effect of urolithiasis and glycyrrhizin on mitochondrial homeostasis / mitochondrial anti-inflammation HaCat cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum. HaCat cells were seeded into 96-well plates with D-gal concentrations of 0, 50, 100, 200, 300, 400, and 500 mM, respectively. After 48 h of treatment, the medium was changed and 10% CCK-8 reagent was added. OD was measured 1 h later. 450 And calculate cell viability and IC50. 30 The experiment was set up with 3 replicates, and the results showed that the IC50 of D-gal was... 30 The initial concentration was 256.26 mM, and 250 mM was selected as the model induction concentration for subsequent experiments.

[0193] Take HaCat cells in the logarithmic growth phase, and after they reach 50% confluence, change the medium and perform the following treatments: (1) NC group: No special treatment; (2) D-gal group: Add 250 mM D-gal; (3) D-gal+UA group: 250 mM D-gal and 5 μM urolithiasis A were added simultaneously; (4) D-gal + UC group: 250 mM D-gal and 5 μM urolithin C were added simultaneously; (5) D-gal + GLD group: 250 mM D-gal and 5 μM glycyrrhizin were added simultaneously; (6) D-gal+UA+GLD group: 250 mM D-gal, 5 μM urolithin A and 5 μM glycyrrhizin were added simultaneously; (7) D-gal+ UC+ GLD group: 250 mM D-gal, 5 μM urolithin C and 5 μM glycyrrhizin were added at the same time.

[0194] After incubation at 37°C and 5% CO2 for 48 hours, cells were collected and mitochondrial membrane potential was labeled using the JC-1 fluorescent probe (Beyotime, C2006) according to the kit instructions.

[0195] The results were tested using one-way ANOVA to determine the significance of differences between groups. P < 0.05 was considered to indicate a significant difference. The highest single agent (HSA) model, a common model for drug combination therapy, was used to further confirm the synergistic trend of the combination.

[0196] D-gal induces a decrease in mitochondrial membrane potential in HaCat cells. The purpose of drug treatment is to inhibit this decrease and increase the membrane potential. The HSA model is a commonly used computational model for drug combination therapy. Therefore, this experiment supplemented the one-way ANOVA with an increasing HSA model to determine the synergistic effect of the combined treatment.

[0197] The formula for the elevated maximum single-drug effect (HSA) model is as follows: ΔHSA = E AB -max(E A E B ) Among them, E A E B E represents the mean values ​​of the two single-drug intervention groups. AB This represents the mean values ​​of the indicators in the joint intervention group.

[0198] When ΔHSA>0, it means E AB The result was higher than the best single-drug group among the two single-drug groups, indicating that the combined intervention was superior to either single intervention and showed a trend of synergistic effect.

[0199] from Figure 2The results showed that, compared with the untreated group (NC), D-gal treatment led to a decrease in mitochondrial membrane potential, manifested as an increase in the proportion of JC-1 monomers and a decrease in aggregates, with a significantly reduced JC-1 aggregate / monomer ratio (P<0.0001). All sample treatments significantly improved this phenomenon, restoring the aggregate / monomer ratio to near-normal levels (P<0.0001). The combination of urolithin A or urolithin C with glycyrrhizin significantly restored the mitochondrial membrane potential decrease caused by oxidative damage, with the combined group showing a significantly better repair effect than the single-component intervention.

[0200] Further analysis using an elevated maximum single-drug effect model revealed that the ΔHSA values ​​for urolithin A combined with glycyrrhizin and urolithin C combined with glycyrrhizin were 5.50 and 7.36, respectively, both greater than 0. This confirms that urolithin combined with glycyrrhizin can synergistically maintain mitochondrial structural and functional homeostasis, thereby inhibiting mitochondrial inflammatory responses.

[0201] Experiment Example 3: Synergistic effect of urolithiasis and glycyrrhizin on skin whitening B16-F10 cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum at 37°C, 5% CO2, and 70% humidity. During the experiment, B16-F10 cells in the logarithmic growth phase were harvested and the medium was changed after reaching 50% confluence. Three replicates were performed, and the following treatments were applied: (1) NC group: No special treatment; (2) Positive control BTR group: 5 μM 4-butylresorcinol; (3) UA group: 5 μM urolithiasis A; (4) UC group: 5 μM urolithiasis C; (5) GLD group: 5 μM glycyrrhizin; (6) UA+GLD group: 5 μM urolithin A and 5 μM glycyrrhizin were added simultaneously; (7) UC+GLD group: 5 μM urolithin C and 5 μM glycyrrhizin were added simultaneously; Cells were collected after co-incubation at 37°C and 5% CO2 for 48 hours for subsequent experiments.

[0202] Tyrosinase activity assay: Tyrosinase activity was detected using a tyrosinase activity assay kit (Solepro, BC4055), and cell protein content was calibrated using a BCA kit (Beyotime, P0012).

[0203] Melanin content detection: Add 300 μL melanin lysis buffer (1 mol / L NaOH containing 10% DMSO), incubate at 80°C for 60 min, centrifuge at 4000×g for 10 min, take the supernatant and detect the absorbance at 475 nm; determine the cell protein content for calibration, and calculate the relative melanin content.

[0204] The results were tested using one-way ANOVA to determine the significance of differences between groups. P < 0.05 was considered to indicate a significant difference. The highest single agent (HSA) model, a common model for drug combination therapy, was used to further confirm the synergistic trend of the combination.

[0205] The purpose of the drug treatment was to reduce tyrosinase activity and melanin content. Therefore, this experiment used a reduction-type Highest Single-Drug Effect (HSA) model in addition to one-way ANOVA to determine the synergistic effect of the combined treatment. The formula for the reduction-type Highest Single-Drug Effect (HSA) model is as follows: ΔHSA=min(E A E B )-E AB Among them, E A E B E represents the mean values ​​of the two single-drug intervention groups. AB This represents the mean values ​​of the indicators in the joint intervention group.

[0206] When ΔHSA>0, it means E AB The result was lower than the best single-drug group among the two single-drug groups, indicating that the combined intervention was superior to either single intervention and showed a trend of synergistic effect.

[0207] The results are as follows Figure 3 As shown, urolithin A or urolithin C alone can significantly inhibit melanin production and tyrosinase activity (P values ​​are P<0.001 and P<0.05, respectively). The combined use of urolithin A and glycyrrhizin, and the combined use of urolithin C and glycyrrhizin, significantly and synergistically enhance the activity (both P<0.0001), and the degree of inhibition by combination is more obvious than that of single drugs.

[0208] Further calculations using the reduced maximum single-drug effect model yielded: (1) Inhibition of tyrosine activity: The ΔHSA of urolithin A combined with glycyrrhizin and urolithin C combined with glycyrrhizin were 0.0026 and 0.0020, respectively, both greater than 0.

[0209] (2) Inhibition of melanin production: The ΔHSA of urolithin A combined with glycyrrhizin and urolithin C combined with glycyrrhizin were 14.51 and 9.33, respectively, both greater than 0.

[0210] This demonstrates that urolithiasis and glycyrrhizin have a synergistic effect on skin whitening.

[0211] Experimental Example 4: Combined application of urolithiasis and glycyrrhizin in skin care I. Experimental Materials

[0212] 1. Test substance Example 1: A skin care cream sample prepared in Comparative Example 1.

[0213] 2. Test instruments Table 12 VISIA-CRImage-ProPlus 7.0 facial image analysis system, IPP image analysis software Percentage of red area on skin, skin ITA° The smaller the percentage of red area measured, the smaller the area of ​​redness; the larger the ITA° measured value, the better the skin tone improvement effect. Tewameter TMHex transepidermal moisture loss test probe Transepidermal water loss (TEWL) value The smaller the measured value, the less transepidermal water loss from the skin, and the better the skin barrier condition. Corneometer CM825 probe for testing stratum corneum moisture content Skin stratum corneum moisture content The higher the measured value, the higher the water content of the stratum corneum of the skin. 3. Subjects Thirty-four female subjects aged 26 to 60 years were selected (32 of whom were valid cases). They were required to be in good general health, without any major systemic diseases, have sensitive skin, and have a positive lactic acid stinging test.

[0214] II. Experimental Methods

[0215] After cleansing their face in the morning and evening, the subjects applied an appropriate amount of the cream sample evenly to their face and gently massaged it in. Apply until fully absorbed, twice daily for 28 consecutive days. Skin parameters were measured on the first day of the trial (D0, baseline), on day 14 of use (D14), and on day 28 of use (D28). The specific measurement methods are as follows: 1. After facial cleansing for 30 minutes, the percentage of red area on the subject's facial skin, ITA°, TEWL value, heme EI value, and stratum corneum moisture content were measured using the above-mentioned testing instruments, and the test data at each time point were recorded.

[0216] 2. During follow-up visits on days 14 and 28, subjects evaluated their facial appearance using a mirror under natural light conditions in the human efficacy laboratory. A 7-point rating system was used to rate and record user satisfaction, with a score of ≥5 indicating a positive evaluation.

[0217] III. Experimental Results 1. Moisturizing function test (1) Water content of the stratum corneum The stratum corneum moisture content of the subjects' skin after using the samples of Example 1 and Comparative Example 1 is shown in Table 13.

[0218] Table 13

[0219] (2) Transepidermal water loss (TEWL) value The transepidermal water loss (TEWL) values ​​of the subjects' skin after using the samples of Example 1 and Comparative Example 1 are shown in Table 16.

[0220] Table 14

[0221] Please refer to Table 13. Compared with D0, the stratum corneum moisture content of the skin showed an increasing trend after the subjects used the samples of Example 1 and Comparative Example 1. Specifically, after using the white cream sample of Comparative Example 1 for 14 days and 28 days, the stratum corneum moisture content increased by 20.43% and 28.31%, respectively. However, after using the white cream sample of Example 1 for 14 days and 28 days, the stratum corneum moisture content increased by 56.89% and 96.88%, respectively, significantly higher than that of the sample of Comparative Example 1, and this effect far exceeded expectations.

[0222] Please refer to Table 14. Compared with D0, the transepidermal water loss (TEWL) values ​​of the skin showed a decreasing trend after the subjects used the samples of Example 1 and Comparative Example 1. Specifically, after using the white cream sample of Comparative Example 1 for 28 days, the transepidermal water loss (TEWL) value of the skin decreased by 15.47%. After using the white cream sample of Example 1 for 28 days, the transepidermal water loss (TEWL) value of the skin decreased by 19.47%, which was also a greater decrease than that of the sample of Comparative Example 1.

[0223] The above results indicate that when urolithiasis A is used in combination with glycyrrhizin, it can synergistically increase the water content of the stratum corneum and reduce the transepidermal water loss (TEWL) value, thereby improving the skin barrier function and enhancing the skin's ability to retain moisture.

[0224] 2. Steady-state / soothing and repair function test (1) Analysis of the red area of ​​the skin The skin redness area values ​​of the subjects after using the samples of Example 1 and Comparative Example 1 are shown in Table 15.

[0225] Table 15

[0226] Please refer to Table 15. Compared to D0, the proportion of the skin red area decreased after subjects used the samples of Example 1 and Comparative Example 1. Specifically, after using the white cream sample of Comparative Example 1 for 14 and 28 days, the proportion of the skin red area decreased by 18.72% and 22.96%, respectively. However, after using the white cream sample of Example 1 for 14 and 28 days, the proportion of the skin red area decreased by 22.56% and 28.55%, respectively, a greater decrease than that of the sample of Comparative Example 1.

[0227] The above results indicate that when urolithin A is used in combination with glycyrrhizin, it produces a synergistic effect in soothing and repairing functions, and significantly improves skin redness.

[0228] 3. Whitening Function Test The skin ITA values ​​of the subjects after using the sample from Example 1 are shown in Table 16.

[0229] Table 16

[0230] Please refer to Table 16. Compared to D0, the skin ITA values ​​of subjects using the sample of Example 1 showed an increasing trend. After 28 days of using the white cream sample of Example 1, the skin ITA value increased by 1.24%.

[0231] The above results demonstrate that the white cream product provided in Example 1, through the multi-dimensional synergistic effect of urolithin A and glycyrrhizin in moisturizing, homeostasis / soothing repair, and whitening, creates a positive cycle of moisturizing, homeostasis / soothing repair, and whitening, ultimately achieving a comprehensive skin condition of "stable, hydrated, and radiant".

[0232] Experimental Example 5: Synergistic effect of retinol in the combination of urolithiasis and glycyrrhizin in an acute oxidative inflammation HaCaT cell model. Experimental procedure: HaCat cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum. Cells were seeded into 96-well plates with H2O2 concentrations of 0, 250, 500, 750, 1000, 1250, 1500, and 2000 μM, respectively. After 2 h of treatment, the medium was changed and 10% CCK-8 reagent was added. OD was measured 2 h later. 450 And calculate cell viability and IC50. 30 The experiment was set up with 3 replicates, and the IC50 of H2O2 was obtained. 20 The initial concentration was 578.08 μM, and 600 μM was selected as the model induction concentration for subsequent experiments.

[0233] Log-phase HaCaT cells were seeded into 6-well plates. After reaching 50% confluence, the medium was changed. The experiment was performed in triplicate, and the following treatment was applied (HaCat cell anti-acute oxidation model): ①NC group: After normal culture for 24 h, the medium was changed to serum-free medium; ②H2O2 group: After normal culture for 24 h, 600 μM H2O2 was added; ③Ret group: After treatment with 12.5 μM retinol for 24 h, 600 μM H2O2 was added; ④UA+ Ret group: After treatment with 5 μM urolithiasis A and 12.5 μM retinol for 24 h, 600 μM H2O2 was added. ⑤GLD+ Ret group: After treatment with 5 μM glycyrrhizin and 12.5 μM retinol for 24 h, 600 μM H2O2 was added; ⑥UA+GLD+Ret group: After treatment with 5 μM urolithiasis A, 5 μM glycyrrhizin and 12.5 μM retinol for 24 h, 600 μM H2O2 was added.

[0234] Cells were incubated at 37℃ and 5% CO2 for 2 h. Total RNA was extracted from each group. The relative expression levels of inflammatory genes (IL-1β, IL-6, TNF-α), aging gene (CDKN1A), stress keratinization gene (KRT16), and collagen degradation gene (MMP1) were detected by qPCR (2^-ΔΔCt method, with β-actin as the internal control). The experiment was set up in 3 replicates to preliminarily evaluate the synergistic effect of urolithin and glycyrrhizin in the anti-inflammatory effect of retinol.

[0235] The results were tested using one-way ANOVA to determine the significance of differences between groups. P < 0.05 was considered to indicate a significant difference. The highest single agent (HSA) model, a common model for drug combination therapy, was used to further confirm the synergistic trend of the combination.

[0236] The purpose of drug treatment is to reduce the expression levels of inflammatory factors and aging genes. Therefore, this experiment used a reduction-type HSA model in addition to one-way ANOVA to determine the synergistic effect of the combined treatment.

[0237] The reduced highest single-drug effect (HSA) model is shown in the following formula: ΔHSA=min(E A E B )-E AB Among them, E A E B E represents the mean values ​​of the two single-drug intervention groups. AB This represents the mean values ​​of the indicators in the joint intervention group.

[0238] When ΔHSA>0, it means E AB The result was lower than the best single-drug group among the two single-drug groups, indicating that the combined intervention was superior to either single intervention and showed a trend of synergistic effect.

[0239] Experimental results: The results are as follows Figure 4As shown, H2O2 treatment significantly upregulated the expression levels of IL-1β, IL-6, TNF-α, CDKN1A, KRT16, and MMP1 in HaCaT cells (P<0.0001), indicating that the cells suffered oxidative damage and triggered inflammation, aging, stress injury, and collagen loss. Compared with the model group, after protective intervention with retinol, the expression levels of most genes were significantly decreased (CDKN1A (P<0.05), IL-6 (P<0.01), KRT16 (P<0.01), MMP1 (P<0.01)), and the expression of IL-1β and TNF-α genes were also downregulated to some extent. However, urolithin A or glycyrrhizin alone did not show a significant synergistic effect of retinol on the expression of genes related to inflammation, aging, and collagen in the inflammatory model. However, the combination of urolithin A and glycyrrhizin unexpectedly enhanced the effects of retinol in the aforementioned aspects, significantly downregulating the expression levels of these genes (P<0.0001) to near-normal cell levels. This demonstrates that the combination of urolithin A and glycyrrhizin significantly promotes the anti-inflammatory capacity of retinol.

[0240] Furthermore, using a reduced-maximum single-drug effect model, with the mean expression value of the "urolithiasis A + retinol" gene being E... A The mean expression level of the "glabridin + retinol" gene was E B "Urolithiasis A + Glycyrrhizin + Retinol" is E AB The calculation yields: (1) CDKN1A: Urolithiasis A + Retinol and Glycyrrhizin + Retinol, compared with Urolithiasis A + Glycyrrhizin + Retinol, ΔHSA = 1.07 > 0; (2) IL1β: Urolithiasis A + retinol and glycyrrhizin + retinol, compared with urolithiasis A + glycyrrhizin + retinol, ΔHSA = 4.70 > 0; (3) IL-6: Urolithiasis A + retinol and glycyrrhizin + retinol, compared with urolithiasis A + glycyrrhizin + retinol, ΔHSA = 2.70 > 0; (4) KRT16: Urolithin A + retinol and glycyrrhizin + retinol, compared with urolithin A + glycyrrhizin + retinol, ΔHSA = 1.77 > 0; (5) MMP1: Urolithiasis A + Retinol and Glycyrrhizin + Retinol, compared with Urolithiasis A + Glycyrrhizin + Retinol, ΔHSA = 3.49 > 0; (6) TNF-α: Urolithiasis A + retinol and glycyrrhizin + retinol, compared with urolithiasis A + glycyrrhizin + retinol, ΔHSA = 3.76 > 0.

[0241] This indicates that the combination of urolithiasis A and glycyrrhizin has a significant synergistic effect on the anti-inflammatory, anti-aging, and collagen loss prevention properties of retinol.

[0242] Experimental Example 6: Synergistic effect of urolithiasis and glycyrrhizin in the action of retinol on the RAR pathway. Experimental procedure: HaCat cells were treated according to the above experimental method (HaCat cell anti-acute oxidation model), with 3 replicates per group, and the groups were as follows: ①NC group: After normal culture for 24 h, the medium was changed to serum-free medium; ②Ret group: After treatment with 12.5 μM retinol for 24 h, serum-free culture medium was added; ③H2O2+Ret group: After treatment with 12.5 μM retinol for 24 h, 600 μM H2O2 was added; ④H2O2+Ret+UA group: After treatment with 12.5μM retinol and 5μM urolithiasis A for 24 h, 600 μM H2O2 was added. ⑤H2O2+Ret+GLD group: After treatment with 12.5μM retinol and 5μM glycyrrhizin for 24 h, 600 μM H2O2 was added; ⑥H2O2+Ret+UA+GLD group: After treatment with 12.5μM retinol, 5 μM urolithiasis A and 5 μM glycyrrhizin for 24 h, 600 μM H2O2 was added. ⑦ H2O2+Ret+UA++GLD+GW9662 group: After treatment with 12.5 μM retinol, 5 μM urolithiasis A, 5 μM glycyrrhizin and 10 μM GW9662 for 24 h, 600 μM H2O2 was added. ⑧H2O2+Ret+BAY 11-7082 group: After treatment with 12.5 μM retinol and 10 μM BAY 11-7082 for 24 h, 600 μM H2O2 was added.

[0243] Among them, GW9662 is a PPARγ antagonist, and BAY 11-7082 is a classic NF-κB pathway inhibitor.

[0244] The cells were incubated at 37℃ and 5% CO2 for 2 h. The total RNA of each group was collected and extracted. The expression levels of CYP26A1 and CRABP2 in each group were detected by qPCR (2^-ΔΔCt method, with β-actin as the internal reference).

[0245] The results were tested using one-way ANOVA to determine the significance of differences between groups. P < 0.05 was considered to indicate a significant difference. The highest single agent (HSA) model, a common model for drug combination therapy, was used to further confirm the synergistic trend of the combination.

[0246] Retinol can indirectly activate the RAR pathway, but this activation is inhibited under inflammatory conditions. The aim of this experiment was to verify that individual drugs and combinations could restore this inhibitory effect and enhance RAR pathway transcription. Therefore, this experiment used an elevated HSA model in addition to one-way ANOVA to determine the synergistic effect of combined treatments.

[0247] The formula for the elevated maximum single-drug effect (HSA) model is as follows: ΔHSA = E AB - max(E A E B ) Among them, E A E B E represents the mean values ​​of the two single-drug intervention groups. AB This represents the mean values ​​of the indicators in the joint intervention group.

[0248] When ΔHSA>0, it means E AB The result was lower than the best single-drug group among the two single-drug groups, indicating that the combined intervention was superior to either single intervention and showed a trend of synergistic effect.

[0249] Experimental results: from Figure 5 As can be seen, compared with normal cells (NC group), retinol significantly increased the expression levels of CYP26A1 and CRABP2 in HaCaT keratinocytes (both P < 0.0001). After hydrogen peroxide stimulation, the transcriptional induction of this gene by retinol (Ret) was significantly reduced (P < 0.0001) to below the level of normal cells, indicating that oxidative stimulation inhibited the retinol-activated RAR pathway transcription. Adding urolithin A or glycyrrhizin alone to retinol (Ret) treatment could restore the expression of both genes to some extent; however, the simultaneous addition of urolithin A and glycyrrhizin could almost completely restore the pro-expression effect of retinol on this gene under hydrogen peroxide stimulation (Ret group vs. H2O2+UA+GLD+Ret group, P > 0.05).

[0250] Further evidence of synergistic effect was obtained using an elevated single-drug effect model, with the mean expression level of the "uriculin A + retinol" gene being E. A The mean expression level of the "glabridin + retinol" gene was E B The mean expression level of the "urolithiasis A + glycyrrhizin + retinol" gene was E. ABThe calculation yields: (1) CYP26A1: Urolithiasis A + Retinol and Glycyrrhizin + Retinol, compared with Urolithiasis A + Glycyrrhizin + Retinol, ΔHSA = 1.41 > 0; (2) CRABP2: Urolithiasis A + Retinol and Glycyrrhizin + Retinol, compared with Urolithiasis A + Glycyrrhizin + Retinol, ΔHSA = 1.37 > 0.

[0251] This demonstrates that the combination of urolithin A and glycyrrhizin can synergistically enhance / restore the promoting effect of retinol on CYP26A1 and CRABP2 gene expression under peroxidative stimulation. This may indicate that the combination of urolithin A and glycyrrhizin can exert a synergistic effect not only in retinol but also in RAR agonists.

[0252] Furthermore, by replacing urolithin A with the conventional NF-κB inhibitor BAY 11-7082 as a promoter to restore the expression of retinol Ret in both genes during peroxidation, the restoration effect was significantly lower than that of urolithin A (P<0.05). This result also indicates that urolithin A has a superior ability to unblock the oxidative inhibition of retinol-enhanced RAR pathway transcription compared to conventional NF-κB inhibitors.

[0253] Furthermore, the addition of GW9662 to group 6 (H2O2+UA+GLD+Ret) revealed that the restorative effect could be partially blocked by the PPARγ antagonist GW9662 (P<0.0001). This may suggest to the inventors that glycyrrhizin contributes to the PPARγ / RXR pathway, thereby synergistically enhancing the retinol effect with urolithin A.

[0254] Experimental Example 7: The synergistic effect of urolithiasis combined with glycyrrhizin on adapalene in an acute inflammatory HaCat cell model. The above experiments suggest that the combination of urolithin A and glycyrrhizin can exert a synergistic effect not only in retinol but also in RAR agonists. This experiment introduces adapalene, an agonist with direct RAR activation, to verify the universality of the RAR agonist effect promoted by the combination of urolithin A and glycyrrhizin.

[0255] Experimental procedure: HaCat cells were treated using the above experimental method (HaCat cell anti-acute oxidation model), with 3 replicates and the following groups: ①NC group: After normal culture for 24 h, the medium was changed to serum-free medium; ②Adapalene: After treatment with 0.1 μM adapalene for 24 h, serum-free culture medium was added; ③H2O2+Adapalene: After treatment with 0.1 μM adapalene for 24 h, 600 μM H2O2 was added; ④ H2O2+Adapalene+UA+GLD group: After treatment with 0.1 μM adapalene, 5 μM urolithiasis A and 5 μM glycyrrhizin for 24 h, 600 μM H2O2 was added.

[0256] Cells were incubated at 37℃ and 5% CO2 for 2 h. Total RNA was extracted from each group. The expression level of CRABP2 in each group was detected by qPCR (2^-ΔΔCt method, with β-actin as the internal reference). The results were analyzed using one-way ANOVA to test the significance of differences between groups. P<0.05 was considered to be significant.

[0257] Experimental results: from Figure 6 As can be seen, similar to retinol, adapalene also significantly increased the expression level of CRABP2 in HaCaT keratinocytes (P<0.0001). After stimulation with hydrogen peroxide, the transcriptional induction of this gene by adapalene was significantly reduced (P<0.0001), while the addition of urolithin A and glycyrrhizin could almost completely restore the pro-expression effect of adapalene on this gene under hydrogen peroxide stimulation (P<0.0001).

[0258] Meanwhile, the expression levels of the CRABP2 gene were compared under hydrogen peroxide induction. In the previous experiment, (retinol + urolithin A + glycyrrhizin) / retinol = 3.22; in this experiment, (adapalene + urolithin A + glycyrrhizin) / adapalene = 2.91, revealing that the recovery ratios were similar or showed the same trend. In summary, the urolithin A + glycyrrhizin combination can achieve significant synergistic effects on both retinol and adapalene, indicating that the synergistic effect of the urolithin A + glycyrrhizin combination on RAR agonists is universal.

[0259] Experimental Example 8: Synergistic Effect of Urolithiasis-Glycyrrhizin Combined with Retinol in a Chronic Oxidative Inflammatory HSF Cell Model Experimental procedure: Human skin fibroblasts (HSF) were cultured in DMEM high-glucose medium containing 10% fetal bovine serum. HSF cells were seeded into 96-well plates with D-gal concentrations of 0, 50, 100, 200, 300, 400, and 500 mM, respectively. After 48 h of treatment, the medium was changed and 10% CCK-8 reagent was added. OD was measured 2 h later. 450And calculate cell viability and IC50. 30 The experiment was set up with 3 replicates, and the results showed that the IC50 of D-gal was... 30 The initial concentration was 152.15 mM, and 150 mM was selected as the model induction concentration for subsequent experiments.

[0260] HSF cells in the logarithmic growth phase were cultured in 6-well plates. After reaching 50% confluence, the medium was changed (HSF cells were subjected to chronic oxidation treatment to establish a model). Three replicates were set up, and the cells were grouped as follows: ①NC group: No special treatment; ② D-gal group: Add 150 mM D-gal; ③ UA group: 150 mM D-gal and 5 μM urolithiasis A were added simultaneously; ④GLD group: 150 mM D-gal and 5 μM glycyrrhizin were added simultaneously; ⑤Ret group: 150 mM D-gal and 50 μM retinol were added simultaneously; ⑥UA+ Ret group: 150 mM D-gal, 5 μM urolithiasis A, and 5 μM retinol were added simultaneously; ⑦GLD+ Ret group: 150 mM D-gal, 5 μM glycyrrhizin, and 5 μM retinol were added simultaneously; ⑧UA+GLD: Simultaneously add 150 mM D-gal, 5 μM urolithin A, and 5 μM glycyrrhizin; ⑨ UA+GLD+Ret group: 150 mM D-gal, 5 μM urolithiasis A, 5 μM glycyrrhizin, and 5 μM retinol were added simultaneously.

[0261] The cells were incubated at 37℃ and 5% CO2 for 48 h. After treatment, cells from each group were collected and total RNA was extracted. The expression level of the inflammatory gene IL-6 was detected by qPCR (2^-ΔΔCt method, with β-actin as the internal control). The experiment was set up in triplicate to preliminarily evaluate the synergistic effect of the combined treatment.

[0262] The results were tested using one-way ANOVA to determine the significance of differences between groups. P < 0.05 was considered to indicate a significant difference. The highest single agent (HSA) model, a common model for drug combination therapy, was used to further confirm the synergistic trend of the combination.

[0263] The purpose of drug treatment is to reduce the expression level of inflammatory factors. Therefore, this experiment used a reduction-type HSA model in addition to one-way ANOVA to determine the synergistic effect of the combined treatment.

[0264] The formula for calculating the reduced highest single-drug effect (HSA) model is as follows: ΔHSA=min(E A E B )-E AB Among them, E A E B E represents the mean values ​​of the two single-drug intervention groups. AB This represents the mean values ​​of the indicators in the joint intervention group.

[0265] When ΔHSA>0, it means E AB The result was lower than the best single-drug group among the two single-drug groups, indicating that the combined intervention was superior to either single intervention and showed a trend of synergistic effect.

[0266] Experimental results: The results are as follows Figure 7 As shown, D-gal treatment significantly upregulated the expression level of IL-6 in HSF cells (P<0.0001), indicating that the cells suffered oxidative damage and the model was successful. Compared with the model group, the expression of IL-6 in the urolithin A group was significantly reduced (P<0.05). The expression levels of IL-6 in the glycyrrhizin and retinol treatment groups were downregulated but not significantly. The combination of urolithin A with glycyrrhizin, urolithin A with retinol, and glycyrrhizin with retinol could further downregulate the expression, but the further downregulation was not significant. However, when urolithin A and glycyrrhizin were used in combination with retinol, the downregulation level was significant (P<0.0001).

[0267] Furthermore, using a reduced-maximum single-drug effect model, with the mean expression value of the "urolithiasis A + retinol" gene being E... A The mean expression level of the "glabridin + retinol" gene was E B "Urolithiasis A + Glycyrrhizin + Retinol" is E AB The calculations show that the difference between urolithiasis A + retinol and glycyrrhizin + retinol is ΔHSA = 1.18 > 0.

[0268] This suggests that the combination of urolithiasis and glycyrrhizin has a synergistic effect on the anti-chronic skin inflammation of retinol.

[0269] The above-described embodiments are merely preferred embodiments provided to fully illustrate the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the scope of protection of the present invention. The scope of protection of the present invention is defined by the claims.

Claims

1. A composition, characterized in that, The composition comprises or is composed of the following components: (i) A compound of formula (I) or a precursor thereof; (ii) Glycyrrhizin or its derivatives; In formula (I): A, B, C, and D are each independently selected from H and OH; W, X, and Y are each independently selected from H and OH; and Z is selected from H and OH.

2. The composition according to claim 1, characterized in that, In the composition, the compounds of formula (I) include urolithin A, urolithin B, urolithin C, urolithin D, urolithin E, urolithin G, urolithin M-5, urolithin M-6, urolithin M-7, isourolithin A, isourolithin B, or combinations thereof.

3. The composition according to claim 1, characterized in that, In the composition, the mass ratio of the compound of formula (I) or its precursor to glycyrrhizin or its derivative is (0.1-1000):1, preferably (1-100):1, and more preferably (5-50):

1.

4. The composition according to claim 1, characterized in that, The purity of the compound of formula (I) or its precursor is 10% or more, 60% or more, 90% or more, 94% or more, 95% or more, 98% or more, or 100%; and / or The purity of the glycyrrhizin or its derivatives is 10% or more, 60% or more, 90% or more, 94% or more, 95% or more, 98% or more, or 100%.

5. A product or formulation, characterized in that, The composition comprises any one of claims 1-4.

6. The product or formulation as described in claim 5, characterized in that, The product or formulation is a liquid, solid, or semi-solid product or formulation; or The product or preparation is a topical product or preparation.

7. The product or formulation as described in claim 6, characterized in that, The external products or preparations mentioned include cosmetics.

8. A formulation comprising: (1) A first formulation containing a compound of formula (I) or a precursor thereof; as well as (2) A second preparation containing glycyrrhizin or its derivatives; In formula (I): A, B, C, and D are each independently selected from H and OH; W, X, and Y are each independently selected from H and OH; and Z is selected from H and OH.

9. The use of the composition of any one of claims 1 to 4, the product or formulation of any one of claims 5 to 7, or the formulation combination of claim 8 in improving stress and related outcomes; preferably, in improving oxidative stress and related outcomes; more preferably, in homeostasis / anti-inflammatory effects; even more preferably, in cellular homeostasis / anti-inflammatory effects; even more preferably, in mitochondrial homeostasis / anti-inflammatory effects.

10. The use of the composition according to any one of claims 1 to 4, the product or formulation according to any one of claims 5 to 7, or the formulation combination according to claim 8 in the preparation of skin care formulations.

11. The application as described in claim 10, characterized in that, The skin care includes maintaining skin homeostasis, anti-inflammatory effects, moisturizing, and / or soothing and repairing effects, and / or skin whitening.

12. The application according to claim 10, characterized in that, The composition, product, formulation, combination of formulations or skin care formulation contains 0.01-5% of a compound of formula (I) or a precursor thereof, by weight percentage. And 0.001-1% of glycyrrhizin or its derivatives; Preferably, the skin care formulation contains 0.01-1% by weight of a compound of formula (I) or a precursor thereof; And 0.01-0.1% of glycyrrhizin or its derivatives.

13. The application according to claim 10, characterized in that, The skin care preparation includes topical preparations; preferably, the dosage form of the skin care preparation includes aqueous solutions, emulsions, ointments, creams, powders, oils, tablets, and lyophilized preparations.

14. Use of the composition of any one of claims 1 to 4, the product or formulation of any one of claims 5 to 7, or the formulation combination of claim 8 in the preparation of a product for enhancing the effect of a RAR agonist, preferably, the RAR agonist includes a vitamin A RAR agonist and a non-vitamin A RAR agonist; more preferably, the vitamin A RAR agonist includes retinol, retinaldehyde, retinyl palmitate, retinyl linoleate, retinyl acetate, retinyl propionate, hydroxypinazone retinate, retinyl retinoate, all-trans retinoic acid, adapalene, and trafarotine, and the non-vitamin A RAR agonist includes iris isoflavones.

15. The use according to claim 14, characterized in that, The effects of the enhanced RAR agonist include promoting skin epidermal renewal or regeneration, improving wrinkles, improving skin elasticity, improving skin firmness, improving collagen loss or promoting collagen repair, improving or maintaining skin barrier function, improving biological aging, improving photoaging, improving uneven skin tone, improving acne, improving skin inflammation, and improving the skin matrix.

16. The use according to claim 14 or 15, characterized in that, The effect of the enhanced RAR agonist is in skin inflammation; preferably, the skin inflammation is chronic skin inflammation and / or low-grade skin inflammation; preferably, the skin inflammation is caused by oxidative stress, ultraviolet radiation, aging, barrier damage, microecological imbalance, environmental pollution or aging; more preferably, the oxidative stress includes acute oxidative stress or chronic oxidative stress.

17. The use according to claim 16, characterized in that, The effect of the enhanced RAR agonist is achieved by increasing or restoring transcription efficiency; preferably, the effect of the enhanced RAR agonist is achieved by increasing or restoring the transcription efficiency of the RAR / RXR pathway.

18. A product for skin care, characterized in that, The product contains, by weight percentage, 0.05-1% of a compound of formula (I) or a precursor thereof, 0.001-0.1% of glycyrrhizin or a derivative thereof, and cosmetically acceptable excipients; Preferably, the product includes creams, serums, and face creams.

19. The use of the composition according to any one of claims 1 to 4, the product or formulation according to any one of claims 5 to 7, the formulation combination according to claim 8, and the product according to claim 18 in skin care, characterized in that, The skin care includes skin homeostasis, anti-inflammation, moisturizing, and / or soothing repair, and / or whitening.

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