Effective composition, synergistic composition, product and method with effects of regulating circadian rhythm and delaying skin aging

Abstract

The invention relates to the technical field of skin care products, in particular to a functional composition, a synergistic composition, a product and a method with effects of regulating circadian rhythm and delaying skin aging. The functional composition comprises a first component and a second component, the first component comprises a cistanche deserticola extract and / or a dogwood extract; and the second component is prepared from Salvianan A and / or Schizolaenone C. The invention further discloses a preparation method of the pharmaceutical composition. The functional composition provided by the invention has an improvement effect on the circadian rhythm, and a new solution is provided for improving the circadian rhythm of skin cells and delaying skin aging.

Description

Technical Field

[0001] The present application relates to the technical field of skin care products, and in particular to a functional composition, a synergistic composition, a product and a method for regulating circadian rhythm and delaying skin aging. Background Art

[0002] Most organisms on Earth, including mammals, have the ability to adapt to the external cycle of day and night. This 24-hour cycle that regulates physiological functions (such as sleep and wakefulness, feeding and excretion, and hormone secretion) is called the "circadian rhythm," also known as the "biological clock." Organisms synchronize with the external environment through external cues such as light, temperature, and food, and are able to automatically maintain a 24-hour daily rhythm even in the absence of external stimuli. It is worth noting that in mammals, there are generally two types of circadian clocks: the master clock, located in the suprachiasmatic nucleus of the hypothalamus. Periodic signals from the suprachiasmatic nucleus are transmitted to peripheral tissues via nerves and hormones, inducing a certain periodicity in various physiological functions throughout the body. The other type of clock is the peripheral clock, present in various peripheral tissues of the body. Peripheral clocks in tissues such as the liver and kidneys are not only regulated by the master clock but also independently respond to external and internal cues. At the molecular level, circadian rhythms are regulated through transcriptional-translational feedback loops of specific proteins. Among them, the core circadian mechanism composed of CLOCK, BMAL1, PER1, and CRY1 plays a crucial role in regulating circadian rhythms. Within the cell nucleus, the two transcription factors, CLOCK and BMAL1, combine to form a heterodimer. This heterodimer then binds to the E-box on DNA to regulate the expression of downstream genes, PER1 and CRY1, as well as numerous hormones, cytokines, and enzymes. Therefore, an organism's 24-hour circadian rhythm begins with the binding of the CLOCK and BMAL1 dimer. Skin cells are no exception and are regulated by this 24-hour circadian rhythm. Disruptions in the circadian rhythm of skin cells can lead to skin aging and other problems.

[0003] Currently, how to improve circadian rhythm is a hot topic in current research. In view of this, this application is proposed. Summary of the Invention

[0004] Based on this, one or more embodiments of the present application provide a functional composition, synergistic composition, product, and method for regulating circadian rhythms and delaying skin aging. The technical solutions include the following:

[0005] One or more embodiments of the present application provide a functional composition having the effects of regulating circadian rhythm and delaying skin aging, the functional composition comprising a first component and a second component;

[0006] The first component includes Cistanche deserticola extract and / or Cornus officinalis extract;

[0007] The second component includes Salvianan A and / or Schizolaenone C.

[0008] In some embodiments of the present application, the efficacy composition includes one or more of the following items (1) to (2), and correspondingly includes one or more of items (3) and (4):

[0009] (1) 10μg-160μg Cistanche deserticola extract;

[0010] (2) 0.5 μg-1.6 μg of Cornus officinalis extract;

[0011] (3) 1 μmol-10 μmol Salvianan A; and

[0012] (4) 1μmol-10μmol Schizolaenone C.

[0013] One or more embodiments of the present application provide a synergistic composition for regulating circadian rhythm and delaying skin aging, wherein the synergistic composition comprises Salvianan A and Schizolaenone C.

[0014] In some embodiments of the present application, the synergistic composition comprises Salvianan A and Schizolaenone C in an amount ratio of (1-10) μmol: (1-10) μmol.

[0015] One or more embodiments of the present application provide the use of the functional composition or the synergistic composition in the preparation of products for regulating circadian rhythm and delaying skin aging.

[0016] In some embodiments of the present application, the product increases the expression levels of the CLOCK gene and the BMAL1 gene.

[0017] In some embodiments of the present application, the product reduces the expression levels of the CDKN2A gene and the CDKN1A gene.

[0018] One or more embodiments of the present application provide a product for regulating circadian rhythm and delaying skin aging, wherein the product comprises the functional composition or the synergistic composition; the product is a beauty product.

[0019] In some embodiments of the present application, the product includes one or more of the following items 1) to 2), and correspondingly includes one or more of items 3) and 4):

[0020] 1) 10μg / mL-160μg / mL Cistanche deserticola extract;

[0021] 2) 0.5 μg / mL-1.6 μg / mL Cornus officinalis extract;

[0022] 3) 1μM-10μM Salvianan A; and,

[0023] 4) 1μM-10μM Schizolaenone C.

[0024] One or more embodiments of the present application provide a method for regulating cell circadian rhythm and delaying cell aging, the method comprising the step of contacting the cells to be treated with the efficacy composition, the synergistic composition or the product.

[0025] Compared with traditional technologies, this application has the following beneficial effects:

[0026] The present application provides a specific composition that has an improving effect on circadian rhythm, providing a new solution for improving the circadian rhythm of skin cells and delaying skin aging. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more fully understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present application. Those skilled in the art can also derive other drawings based on these drawings without inventive effort.

[0028] Figure 1 The effects of various intervention schemes on the expression of CLOCK and BMAL1 proteins in HaCaT cells. DETAILED DESCRIPTION

[0029] Below in conjunction with accompanying drawing, embodiment and example, the application is described in further detail.It should be understood that these embodiment and example are only used to illustrate the application and are not used to limit the scope of the application, and the purpose of providing these embodiment and example is to make the understanding of the disclosure of the application more thorough and comprehensive.It should also be understood that the application can be implemented in many different forms and is not limited to the embodiment and example described herein. Those skilled in the art can make various changes or modifications without violating the connotation of the application, and the equivalent form obtained also falls within the protection scope of the application.In addition, in the description below, a large amount of specific details are given in order to provide a more comprehensive understanding of the application.It should be understood that the application can be implemented without one or more of these details.

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application pertains. The terms used herein in the specification of this application are for the purpose of describing embodiments and examples only and are not intended to limit this application.

[0031] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0032] The terms "and / or", "or / and", and "and / or" used herein include any one of two or more related listed items, and also include any and all combinations of the related listed items, wherein the arbitrary and all combinations include any combination of two related listed items, any more related listed items, or all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical and" and also undoubtedly includes technical solutions connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution of all being connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the four-item combination of A, B, C, and D (that is, the technical solution of all being connected by "logical AND").

[0033] In this application, "plurality", "multiple", "multiple times", "multiples", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0034] As used herein, "combination thereof", "any combination thereof", "any combination thereof" and the like include all suitable combinations of any two or more of the listed items.

[0035] Herein, the “suitable” mentioned in “suitable combination”, “suitable method”, “any suitable method”, etc. shall be based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0036] Herein, "preferred", "better", "more preferred" and "suitable" are merely used to describe implementation methods or examples with better effects. It should be understood that they do not constitute limitations on the scope of protection of this application.

[0037] In this application, "further", "further", "particularly" and the like are used for descriptive purposes to indicate differences in content, but should not be understood as limiting the scope of protection of this application.

[0038] In this application, the terms "optionally," "optional," and "optional" mean optional or dispensable, i.e., they refer to either option being selected from two parallel options: "with" or "without." If a technical solution contains multiple "optional" clauses, each "optional" clause is independent unless otherwise specified and there are no contradictions or constraints.

[0039] In this application, the terms "first," "second," "third," "fourth," etc. in "the first aspect," "the second aspect," "the third aspect," "the fourth aspect," etc. are used for descriptive purposes only and should not be understood as indicating or implying relative importance or quantity, nor should they be understood as implicitly indicating the importance or quantity of the indicated technical features. Furthermore, "first," "second," "third," "fourth," etc. serve only as non-exhaustive enumeration and description, and should be understood not to constitute a closed-ended limitation on quantity.

[0040] In this application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0041] In this application, when referring to a numerical interval (i.e., a numerical range), unless otherwise specified, the distribution of the optional values ​​within the numerical interval is considered continuous and includes the two numerical endpoints of the numerical range (i.e., the minimum and maximum values), as well as every numerical value between these two numerical endpoints. Unless otherwise specified, when a numerical interval refers only to integers within the numerical interval, it includes the two numerical endpoints of the numerical range, as well as every integer between the two numerical endpoints. In this document, this is equivalent to directly listing each integer. For example, "t is an integer selected from 1 to 10" means that t is any integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. In addition, when multiple ranges are provided to describe a feature or characteristic, these ranges may be combined. In other words, unless otherwise specified, ranges disclosed herein should be understood to include any and all subranges subsumed therein.

[0042] Unless otherwise specified, the temperature parameters in this application allow for both constant temperature treatment and temperature fluctuations within a certain temperature range. It should be understood that the constant temperature treatment allows for temperature fluctuations within the accuracy range of instrument control. Fluctuations within ranges such as ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are permitted.

[0043] In this application, %(w / w) and wt% both refer to weight percentage, %(v / v) refers to volume percentage, and %(w / v) refers to mass volume percentage.

[0044] All documents mentioned in this application are cited as references in this application, just as each document is cited as reference separately. Unless they conflict with the application purpose and / or technical solution of this application, the cited documents involved in this application are cited in their entirety and for all purposes. When cited documents are involved in this application, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When cited documents are involved in this application, the examples and preferred embodiments of the cited relevant technical features may also be incorporated into this application as references, but are limited to the ability to implement this application. It should be understood that when the cited content conflicts with the description in this application, the present application shall prevail or be adaptively amended according to the description in this application.

[0045] In a first aspect of the embodiments of the present application, a functional composition having the effects of regulating circadian rhythm and delaying skin aging is provided, wherein the functional composition comprises a first component and a second component;

[0046] The first component includes Cistanche deserticola extract and / or Cornus officinalis extract;

[0047] The second component includes Salvianan A and / or Schizolaenone C.

[0048] In some examples of the present application, the effective composition includes a combination of Cistanche deserticola extract and Salvianan A, or a combination of Cornus officinalis extract and Salvianan A, or a combination of Cistanche deserticola extract and Schizolaenone C, or a combination of Cornus officinalis extract and Schizolaenone C, or a combination of Cistanche deserticola extract, Cornus officinalis extract and Salvianan A, or a combination of Cistanche deserticola extract, Cornus officinalis extract and Schizolaenone C, or a combination of Cistanche deserticola extract, Salvianan A and Schizolaenone C, or a combination of Cornus officinalis extract, Salvianan A and Schizolaenone C, or a combination of Cistanche deserticola extract, Cornus officinalis extract, Salvianan A and Schizolaenone C.

[0049] The present application further defines the relationship between the amounts of the first component and the second component, where the first component is characterized by mass and the second component is characterized by molar number.

[0050] In some examples of the present application, the efficacy composition includes one or more of the following items (1) to (2), and correspondingly includes one or more of the following items (3) and (4):

[0051] (1) 10μg-160μg Cistanche deserticola extract;

[0052] (2) 0.5 μg-1.6 μg of Cornus officinalis extract;

[0053] (3) 1 μmol-10 μmol Salvianan A; and

[0054] (4) 1μmol-10μmol Schizolaenone C.

[0055] In some examples of the present application, the efficacy composition includes 10μg-160μg (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160μg) of Cistanche deserticola extract. Under this condition, it correspondingly includes 1μmol-10μmol (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10μmol) of Salvianan A.

[0056] In some examples of the present application, the efficacy composition includes 0.5 μg-1.6 μg (for example, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6 μg) of Cornus officinalis extract. Under this condition, it correspondingly includes 1 μmol-10 μmol (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 μmol) of Salvianan A.

[0057] In some examples of the present application, the efficacy composition includes 10μg-160μg (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160μg) of Cistanche deserticola extract, and under this condition, it correspondingly includes 1μmol-10μmol (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10μmol) of Schizolaenone C.

[0058] In some examples of the present application, the efficacy composition includes 0.5μg-1.6μg (for example, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6μg) of Cornus officinalis extract, and under this condition, it correspondingly includes 1μmol-10μmol (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10μmol) of Schizolaenone C.

[0059] In some examples of the present application, the efficacy composition includes 10μg-160μg (for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160μg) of Cistanche deserticola extract, 0.5μg-1.6μg (for example, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6μg) of Cornus officinalis extract. Under this condition, it correspondingly includes 1μmol-10μmol (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10μmol) of Salvianan A.

[0060] In some examples of the present application, the efficacy composition includes 10μg-160μg (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160μg) of Cistanche deserticola extract, 0.5μg-1.6μg (e.g., 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6μg) of Cornus officinalis extract. Under this condition, it correspondingly includes 1μmol-10μmol (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10μmol) of Schizolaenone C.

[0061] In some examples of the present application, the efficacy composition includes 10μg-160μg (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160μg) of Cistanche deserticola extract, under which conditions, it correspondingly includes 1μmol-10μmol (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10μmol) of Salvianan A and 1μmol-10μmol (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10μmol) of Schizolaenone C.

[0062] In some examples of the present application, the efficacy composition includes 0.5μg-1.6μg (for example, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6μg) of Cornus officinalis extract, which, under these conditions, correspondingly includes 1μmol-10μmol (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10μmol) of Salvianan A and 1μmol-10μmol (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10μmol) of Schizolaenone C.

[0063] In some examples of the present application, the efficacy composition includes 10μg-160μg (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160μg) of Cistanche deserticola extract and 0.5μg-1.6μg of Cornus officinalis extract. Under this condition, it correspondingly includes 1μmol-10μmol (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10μmol) of Salvianan A and 1μmol-10μmol (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10μmol) of Schizolaenone C.

[0064] It is understood that, under the conditions determined by this corresponding relationship, when the content of the first component is increased or decreased, the content of the second component is also increased or decreased accordingly. For example, if the amount of Cistanche deserticola extract in the efficacy composition is increased to 10 mg-160 mg, the amount of Salvianan A in the efficacy composition is correspondingly increased to 1 mmol-10 mmol.

[0065] In a second aspect of the embodiments of the present application, a synergistic composition for regulating circadian rhythm and delaying skin aging is provided, wherein the synergistic composition comprises Salvianan A and Schizolaenone C.

[0066] In some examples of the present application, the synergistic composition comprises Salvianan A and Schizolaenone C in an amount ratio of (1-10) μmol: (1-10) μmol. Salvianan A and Schizolaenone The molar ratio of C is, for example, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 2:1, 2:3, 2:4, 2:5, 2:6, 2:7, 2:8, 2:9, 2:10, 3:1, 3:2, 3:4, 3:5, 3:6, 3:7, 3:8, 3:9, 3:10, 4:1, 4:2, 4:3, 4:5, 4:6, 4:7, 4:8, 4:9, 4:10, 5:1, 5:2, 5:3, 5:4, 5:6, 5:7, 5:8, 5:9, 5:1 0, 6:1, 6:2, 6:3, 6:4, 6:5, 6:7, 6:8, 6:9, 6:10, 7:1, 7:2, 7:3, 7:4, 7:5, 7:6, 7:8, 7:9, 7:10, 8:1, 8:2, 8:3, 8:4, 8:5, 8:6, 8:7, 8:9, 8:10, 9:1, 9:2, 9:3, 9:4, 9:5, 9:6, 9:7, 9:9, 9:10, 10:1, 10:2, 10:3, 10:4, 10:5, 10:6, 10:7, 10:8, 10:9.

[0067] The third aspect of the embodiments of the present application provides the use of the functional composition described in the first aspect or the synergistic composition described in the second aspect in the preparation of a product for regulating circadian rhythm and delaying skin aging.

[0068] In some examples of the present application, the product increases the expression levels of the CLOCK gene and the BMAL1 gene.

[0069] In some examples of the present application, the product reduces the expression levels of the CDKN2A gene and the CDKN1A gene.

[0070] A fourth aspect of the embodiments of the present application provides a product for regulating circadian rhythm and delaying skin aging, wherein the product comprises the functional composition described in the first aspect or the synergistic composition described in the second aspect.

[0071] This application does not specifically limit the type of the circadian rhythm improving product, which may be, for example, food, medicine, or cosmetics.

[0072] In some examples of the present application, the product includes one or more of the following items 1) to 2) and correspondingly includes one or more of items 3) and 4):

[0073] 1) 10μg / mL-160μg / mL Cistanche deserticola extract;

[0074] 2) 0.5 μg / mL-1.6 μg / mL Cornus officinalis extract;

[0075] 3) 1μM-10μM Salvianan A; and,

[0076] 4) 1μM-10μM Schizolaenone C.

[0077] In some examples of the present application, the product includes 10 μg / mL-160 μg / mL (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160 μg / mL) of Cistanche deserticola extract and 1 μmol / mL-10 μmol / mL (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 μmol / mL) of Salvianan A.

[0078] In some examples of the present application, the product includes 0.5 μg / mL-1.6 μg / mL (for example, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6 μg / mL) of Cornus officinalis extract and 1 μmol / mL-10 μmol / mL (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 μmol / mL) of Salvianan A.

[0079] In some examples of the present application, the product includes 10 μg / mL-160 μg / mL (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160 μg / mL) of Cistanche deserticola extract and 1 μmol / mL-10 μmol / mL (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 μmol / mL) of Schizolaenone C.

[0080] In some examples of the present application, the product includes 0.5 μg / mL-1.6 μg / mL (e.g., 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6 μg / mL) of Cornus officinalis extract and 1 μmol / mL-10 μmol / mL (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 μmol / mL) of Schizolaenone C.

[0081] In some examples of the present application, the product includes 10 μg / mL-160 μg / mL (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160 μg / mL) of Cistanche deserticola extract, 0.5 μg / mL-1.6 μg / mL (e.g., 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6 μg / mL) of Cornus officinalis extract, and 1 μmol / mL-10 μmol / mL (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 μmol / mL) of Salvianan A.

[0082] In some examples of the present application, the product includes 10 μg / mL-160 μg / mL (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160 μg / mL) of Cistanche deserticola extract, 0.5 μg / mL-1.6 μg / mL (e.g., 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6 μg / mL) of Cornus officinalis extract, and 1 μmol / mL-10 μmol / mL (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 μmol / mL) of Schizolaenone C.

[0083] In some examples of the present application, the product includes 10 μg / mL-160 μg / mL (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160 μg / mL) of Cistanche deserticola extract, 1 μmol / mL-10 μmol / mL (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 μmol / mL) of Salvianan A and 1 μmol / mL-10 μmol / mL (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 μmol / mL) of Schizolaenone C.

[0084] In some examples of the present application, the product includes 0.5 μg / mL-1.6 μg / mL (for example, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6 μg / mL) of Cornus officinalis extract, 1 μmol / mL-10 μmol / mL (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 μmol / mL) of Salvianan A and 1 μmol / mL-10 μmol / mL (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 μmol / mL) of Schizolaenone C.

[0085] In some examples of the present application, the product includes 10 μg / mL-160 μg / mL (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160 μg / mL) of Cistanche deserticola extract, 0.5 μg / mL-1.6 μg / mL (e.g., 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6 μg / mL) of Cornus officinalis extract, 1 μmol / mL-10 μmol / mL (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 μmol / mL) of Salvia miltiorrhiza extract, A and 1 μmol-10 μmol (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 μmol / mL) schizolaenone C.

[0086] In a fifth aspect of the embodiments of the present application, a method for regulating cell circadian rhythm and delaying cell aging is provided, which comprises the step of contacting the cells to be treated with the efficacy composition, the synergistic composition or the product.

[0087] In a fifth aspect of the embodiments of the present application, a method for regulating cell circadian rhythm and delaying cell aging is provided, which comprises the step of contacting the cells to be treated with the efficacy composition, the synergistic composition or the product.

[0088] The embodiments of the present application will be described in detail below with reference to the examples. It should be understood that these examples are intended to illustrate the present application only and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and can also be based on the experimental manuals or conventional conditions in this area, or according to the conditions recommended by the manufacturer, or with reference to experimental methods known in the art.

[0089] In the following specific examples, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument testing accuracy or operational accuracy are allowed.

[0090] 1. Experimental Reagents

[0091] 1. Human skin keratinocyte HaCaT cells were purchased from Zhejiang Meisen Cell Technology Co., Ltd., China.

[0092] 2. DMEM culture medium and FBS fetal bovine serum were purchased from Gibco, USA.

[0093] 3. Cell Counting Kit (CCK-8) cell proliferation-toxicity detection kit was purchased from Tongren Chemical Research Institute.

[0094] 4. Taq Pro Universal SYBR qPCR Master Mix was purchased from Novozymes Biotech Co., Ltd., Nanjing, China.

[0095] 5. RNA-easy Isolation Reagent was purchased from Novozymes Biotech Co., Ltd., Nanjing, China.

[0096] 6. FastKing one-step genomic cDNA first-strand synthesis premix reagent was purchased from China Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0097] 7. Doxorubicin hydrochloride was purchased from MedChemExpress, USA.

[0098] 8. Salvianan A (CAS: 2162956-63-8) and Schizolaenone C (CAS: 928760-56-9) were purchased from WuXi AppTec.

[0099] 9. Cistanche deserticola extract was purchased from Shanxi Ruisi Biotechnology Co., Ltd.

[0100] 10. Cornus officinalis extract was purchased from Shaanxi Yunhe Biotechnology Co., Ltd.

[0101] All chemical reagents are conventional reagents.

[0102] 2. Experimental Methods and Results

[0103] 2.1 CCK-8 cytotoxicity assay

[0104] The CCK-8 method was used to screen out the concentration of the test substance that had no significant inhibitory effect on the viability of HaCaT cells. The method was as follows:

[0105] (1) HaCaT cells in the logarithmic growth phase were obtained, digested, and prepared into a cell suspension using DMEM complete medium supplemented with 10% (v / v) fetal bovine serum and 1% (v / v) double-antibody. The cells were then inoculated into 96-well plates, with 100 μL of cell suspension in each well and a cell count of 1 × 10 4 Cells were cultured overnight in a 37°C, 5% CO2 incubator. When the cell confluence reached approximately 80%, the test substances were added to the cells, representing the experimental group. The intervention schedule is shown in Table 1 below, where the concentrations are the working concentrations of the test substances. Wells containing only culture medium served as blank groups, while wells containing culture medium (without the test substance) and cells served as controls.

[0106] (2) Each group of cells was placed in a 37°C, 5% CO2 constant temperature incubator for 24 hours. The original culture medium was replaced with a culture medium supplemented with 10% (v / v) CCK-8 reagent (i.e., 10 mL of CCK-8 reagent was added to every 100 mL of DMEM complete culture medium containing 10% fetal bovine serum and 1% double antibody). The 96-well plate was placed in a constant temperature incubator for 4 hours. The OD value of each well was measured at a wavelength of 450 nm. The proliferation and survival rate of the cells affected by the different test substances was calculated according to the formula. Five replicate wells were performed for each concentration.

[0107] Proliferation survival rate (%) = {(OD 实验组 -OD 空白组 ) / (OD 对照组 -0D 空白组 )}×100%.

[0108] (3) The corresponding HaCaT cell survival rates of each group are shown in Table 1. The results showed that the test substances in each group had no significant inhibitory effect on the survival rate of HaCaT cells.

[0109] Table 1

[0110]

[0111] 2.2 Effects on CLOCK and BMAL1 mRNA Expression in HaCaT Cells

[0112] HaCaT cells treated with 3 μM doxorubicin hydrochloride were selected and treated with one or more of the test substances. RNA was extracted and reverse transcribed, and qPCR was performed to evaluate the mRNA expression levels of the CLOCK and BMAL1 genes in HaCaT cells. The method was as follows:

[0113] (1) HaCaT cells in the logarithmic growth phase were digested and prepared into a cell suspension using DMEM complete medium supplemented with 10% (v / v) fetal bovine serum and 1% (v / v) double antibody. 1×10 6HaCaT cells were cultured in 6-cm cell culture dishes, 5 mL per well, in a 37°C, 5% CO2 incubator. After 24 hours of adaptive culture, the control group was replaced with fresh complete medium, the model group was replaced with fresh complete medium containing 3 μM doxorubicin hydrochloride, and the experimental group was replaced with fresh complete medium containing the corresponding test substance and 3 μM doxorubicin hydrochloride. Fresh complete medium refers to DMEM supplemented with 10% (v / v) fetal bovine serum and 1% (v / v) dual-antibody. The intervention schedule for each group is shown in Table 2 below.

[0114] (2) After culturing in a 37°C, 5% CO2 incubator for 24 hours, extract RNA from each cell group according to the RNA-easy Isolation Reagent instructions and measure RNA concentration. Reverse transcribe the extracted RNA from each group into cDNA at a concentration of 100 ng / μL according to the FastKing One-Step Genomic cDNA First-Strand Synthesis Premix. Finally, prepare the experimental system according to the Taq Pro Universal SYBR qPCR Master Mix instructions, and use the housekeeping gene GAPDH as an internal reference to measure the mRNA expression levels of the CLOCK and BMAL1 genes.

[0115] (3) The results are shown in Table 2. The results showed that after HaCaT cells were treated with 3 μM doxorubicin hydrochloride, the mRNA levels of CLOCK and BMAL1 genes were significantly reduced. After the addition of the test substance, the mRNA expression of CLOCK and BMAL1 genes was affected. In addition, the combination of the test substances had a better effect on restoring circadian rhythm imbalance than the single substance.

[0116] Table 2

[0117]

[0118] 2.3 Effects on CLOCK and BMAL1 protein expression in HaCaT cells

[0119] The test substance was selected to intervene in HaCaT cells treated with 3 μM doxorubicin hydrochloride, and the protein was extracted and then subjected to Western blot analysis to evaluate the expression levels of CLOCK and BMAL1 proteins in HaCaT cells. The method was as follows:

[0120] (1) HaCaT cells in the logarithmic growth phase were digested and prepared into a cell suspension using DMEM complete medium supplemented with 10% fetal bovine serum and 1% double antibody. 1×10 6HaCaT cells were cultured in 6-cm cell culture dishes, 5 mL per well, in a 37°C, 5% CO2 incubator. After 24 hours of adaptive culture, the control group was replaced with fresh complete medium, the model group was replaced with fresh complete medium containing 3 μM doxorubicin hydrochloride, and the experimental group was replaced with fresh complete medium containing the test substance and 3 μM doxorubicin hydrochloride. Fresh complete medium refers to DMEM supplemented with 10% (v / v) fetal bovine serum and 1% (v / v) dual-antibody. The intervention schedule for each group is shown in Table 3.

[0121] (2) After culturing in a constant temperature incubator at 37°C and 5% CO2 for 24 h, the cells were scraped off with a cell spatula, centrifuged at 1000 rpm for 5 min, lysed on ice to extract total cell protein, and the protein concentration was determined using a BCA kit. 30 μg of protein from each group was subjected to SDS-PAGE electrophoresis, transferred to a PVDF membrane, blocked with 5% BSA, and incubated overnight on a shaker at 4°C with the addition of primary antibodies for CLOCK, BMAL1, and GAPDH. The membrane was washed with TBS-T washing solution, and HRP-labeled secondary antibody was added and incubated at room temperature for 1 h. The membrane was washed with TBS-T washing solution and photographed using a chemiluminescence imaging system.

[0122] (3) The results are as follows Figure 1 and as shown in Table 3. Figure 1 The results show the effects of the test substances on the expression of CLOCK and BMAL1 proteins in HaCaT cells. "Combination" shows the test results of experimental group 3-13. The results show that after HaCaT cells were treated with 3 μM doxorubicin hydrochloride, the expression of CLOCK and BMAL1 proteins was significantly reduced; after intervention with the test substances, the expression of CLOCK and BMAL1 proteins increased.

[0123] Table 3

[0124]

[0125] 2.4 Effects on mRNA expression of CDKN2A and CDKN1A genes in HaCaT cells

[0126] The test substance was selected to intervene in HaCaT cells treated with 3 μM doxorubicin hydrochloride, RNA was extracted and reverse transcribed, and qPCR experiments were performed to evaluate the mRNA expression levels of CDKN2A and CDKN1A genes in HaCaT cells. The method was as follows:

[0127] (1) HaCaT cells in the logarithmic growth phase were digested and prepared into a cell suspension using DMEM complete medium containing 10% (v / v) fetal bovine serum and 1% double antibody (v / v). 1×10 6Cells were cultured in 6 cm cell culture dishes, 5 mL per well, in a 37°C, 5% CO2 incubator. After 24 hours of adaptive culture, the control group was replaced with fresh complete medium, the model group was replaced with fresh complete medium containing 3 μM doxorubicin hydrochloride, and the experimental group was replaced with fresh complete medium containing the test substance and 3 μM doxorubicin hydrochloride. Fresh complete medium refers to RPMI complete medium supplemented with 10% (v / v) fetal bovine serum and 1% (v / v) dual-antibody. The intervention schedule for each group is shown in Table 4 below.

[0128] (2) After culturing in a 37°C, 5% CO2 incubator for 24 hours, extract RNA from each cell group according to the RNA-easy Isolation Reagent instructions and measure RNA concentration. Reverse transcribe the extracted RNA from each group into cDNA at a concentration of 100 ng / μL according to the FastKing One-Step Genomic cDNA First-Strand Synthesis Premix. Finally, prepare the experimental system according to the Taq Pro Universal SYBR qPCR Master Mix instructions to measure the mRNA expression levels of the CDKN2A and CDKN1A genes.

[0129] (3) The results are shown in Table 4. The results showed that after HaCaT cells were treated with 3 μM doxorubicin hydrochloride, the mRNA expression of CDKN2A and CDKN1A genes increased significantly; after the test substances were added for intervention, the mRNA expression of CDKN2A and CDKN1A decreased.

[0130] Table 4

[0131]

[0132] GraphPad Prism 9.3.0 statistical software was used for data processing, and the experimental results were expressed as Mean ± SD.

[0133] The various technical features of the above-mentioned implementation modes and examples can be combined in any appropriate manner. In order to make the description concise, not all possible combinations of the various technical features in the above-mentioned implementation modes and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of the description in this specification.

[0134] The above-described embodiments only express several implementation methods of the present application, which facilitate a specific and detailed understanding of the technical solutions of the present application, but cannot be understood as limiting the scope of protection of the patent application. It should be pointed out that, for ordinary technicians in this field, without departing from the concept of the present application, several variations and improvements can be made, which all fall within the scope of protection of the present application. In addition, it should be understood that after reading the above-mentioned teaching content of the present application, those skilled in the art can make various changes or modifications to the present application, and the equivalent forms obtained also fall within the scope of protection of the present application. It should also be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided in the present application are all within the scope of protection of the claims attached to the present application. Therefore, the scope of protection of the patent application of the present application shall be based on the content of the attached claims, and the description and drawings can be used to interpret the content of the claims.

Claims

1. A composition having the effects of regulating circadian rhythm and delaying skin aging, characterized in that: The efficacy composition includes a first component and a second component; The first component includes Cistanche deserticola extract and / or Cornus officinalis extract; The second component includes Salvianan A and / or Schizolaenone C.

2. The functional composition having the effects of regulating circadian rhythm and delaying skin aging according to claim 1, characterized in that: The efficacy composition includes one or more of the following items (1) to (2), and correspondingly includes one or more of the following items (3) and (4): (1) 10μg-160μg Cistanche deserticola extract; (2) 0.5 μg-1.6 μg of Cornus officinalis extract; (3) 1 μmol-10 μmol Salvianan A; and (4) 1μmol-10μmol Schizolaenone C.

3. A synergistic composition for regulating circadian rhythm and delaying skin aging, characterized in that: The synergistic combination includes Salvianan A and Schizolaenone C.

4. The synergistic composition for regulating circadian rhythm and delaying skin aging according to claim 3, characterized in that: The synergistic composition comprises Salvianan A and Schizolaenone C in an amount ratio of (1-10) μmol: (1-10) μmol.

5. Use of the functional composition according to any one of claims 1 to 2 or the synergistic composition according to any one of claims 3 to 4 in the preparation of a product for regulating circadian rhythm and delaying skin aging.

6. The use according to claim 5, characterized in that The product increases the expression levels of the CLOCK gene and the BMAL1 gene.

7. The use according to any one of claims 5 to 6, characterized in that The product reduces the expression levels of the CDKN2A gene and the CDKN1A gene.

8. A beauty product for regulating circadian rhythm and delaying skin aging, characterized in that: The product comprises the efficacy composition according to any one of claims 1 to 2 or the synergistic composition according to any one of claims 3 to 4.

9. The cosmetic product for regulating circadian rhythm and delaying skin aging according to claim 8, characterized in that: The products include one or more of the following items 1) to 2) and one or more of the following items 3) and 4) respectively: 1) 10μg / mL-160μg / mL Cistanche deserticola extract; 2) 0.5 μg / mL-1.6 μg / mL Cornus officinalis extract; 3) 1μM-10μM Salvianan A; and, 4) 1μM-10μM Schizolaenone C.

10. A method for regulating cell circadian rhythm and delaying cell aging, characterized in that: The method comprises the step of contacting the cells to be treated with the efficacy composition of any one of claims 1 to 2, the synergistic composition of any one of claims 3 to 4, or the product of any one of claims 8 to 9.

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