CN116440035A - Regulator of biological rhythm gene expression quantity, application thereof, skin care product and cosmetics - Google Patents
Regulator of biological rhythm gene expression quantity, application thereof, skin care product and cosmetics Download PDFInfo
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- CN116440035A CN116440035A CN202310338316.0A CN202310338316A CN116440035A CN 116440035 A CN116440035 A CN 116440035A CN 202310338316 A CN202310338316 A CN 202310338316A CN 116440035 A CN116440035 A CN 116440035A
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Classifications
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Landscapes
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Abstract
The invention discloses a regulator of biological rhythm gene expression quantity, which comprises a promoter, wherein the promoter comprises a skin conditioner A mainly composed of glycoprotein and amino acid, a skin conditioner B containing glutamyl amino ethyl imidazole and adenosine; the regulator further comprises an enhancer comprising a lespedeza extract. The invention also provides application of the regulator of the biological rhythm gene expression level in skin care products and cosmetics. The invention also provides a skin care product and a cosmetic. The regulator of the biological rhythm gene expression level comprises the accelerator and the lespedeza extract enhancer, and can efficiently promote the expression of the accelerator on the CLOCK gene and the Per1 gene by the interaction of the lespedeza extract and the accelerator, thereby reducing the damage of skin DNA and promoting the repair of cell scratch damage.
Description
Technical Field
The invention belongs to the field of skin care products, and particularly relates to a gene expression regulator, application thereof, a skin care product and a cosmetic.
Background
Circadian Rhythms (CR), also known as biological clock, refer to the activities of sleep, wakefulness, eating, etc., of an organism, as well as the rhythmic changes of various physiological, biochemical, metabolic processes, following approximately 24 hours. The biological clock consists of various peripheral tissue biological clocks located in the upper nucleus of hypothalamic visual intersection and in vivo, and the biological clock is circulated periodically by day and night change of the earth for 24 hours. Circadian rhythms are widely available in many species, and are affected by the earth's circadian variation, a result of biological adaptation to the earth's environment. There is also an independent biological clock system in keratinocytes, melanocytes, and fibroblasts, which are skin cells. The biological clock proteins are expressed not only in cultured human cells but also in human skin biological tissues and thereby have an effect on skin function.
The molecular mechanism of biological clocks is currently most accepted as the positive and negative feedback loop mechanism of biological rhythms. From a molecular perspective, biological rhythms are conserved transcriptional-translational feedback loops consisting of a complete set of positive and negative transcriptional regulatory elements. The heteroduplex formed by the nuclear protein molecules Bmal1 and CLOCK induces the expression of genes such as E-box promoter gene and Pers, crys, rev-Erb. And after Pers and Crys accumulate to a certain degree, heterodimers are formed and the activity of Bmal1/CLOCK is re-inhibited, so that the transcription and translation of Pers and Crys are reduced, and a negative feedback loop is formed. Periodic rhythms of gene expression associated with biological clocks are generated by transcription-translation feedback loops of biological rhythmic proteins. Among them, the CLOCK gene and the Pers gene exert very important functions.
At present, a few researches are focused on efficacy raw materials or compositions for influencing the circadian rhythm of skin, for example, patent CN110448478A provides a composition and application thereof in preparing skin care products for regulating the circadian rhythm of skin, the patent selects skin conditioner A containing glycoprotein and amino acid, skin conditioner B containing glutamyl amino ethyl imidazole and adenosine as raw materials to prepare the composition with the function of regulating the circadian rhythm of skin, the composition mainly researches a series of problems for promoting cell energy synthesis to improve the skin of a night-user in a molecular mechanism level, only simply mentions that the expression of biological CLOCK genes can be regulated, does not carry out intensive research on the expression of the biological CLOCK genes, and further researches show that the composition has a certain promoting effect on the expression of the biological CLOCK genes, but has limited promoting effect, and especially has a promoting effect on the expression of CLOCK genes and Per1 genes.
Disclosure of Invention
The invention aims to overcome the defects and the shortcomings in the background art, and provides a regulator of the biological rhythm gene expression level with good effect of promoting the expression of a CLOCK gene and a Per1 gene, and application of the regulator in skin care products or cosmetics, and skin care products and cosmetics. In order to solve the technical problems, the technical scheme provided by the invention is as follows:
a regulator of biological rhythm gene expression quantity, comprising a promoter, wherein the promoter comprises a skin conditioner A mainly composed of glycoprotein and amino acid, a skin conditioner B containing glutamylaminoethyl imidazole and adenosine; the regulator further comprises an enhancer comprising a lespedeza extract.
In the above regulator, preferably, the amount of the Lespedeza extract is 0.02-10 times of the mass of the promoter.
In the above regulator, preferably, the amount of the Lespedeza extract is 0.2-6 times of the mass of the promoter.
In the above regulator, preferably, the lespedeza extract is a solution obtained by dissolving lespedeza extract powder in propylene glycol-water solution, wherein the mass content of the lespedeza extract powder is 0.1-10%, and the mass content of propylene glycol is 30-50%.
In the above regulator, preferably, the preparation method of the lespedeza extract comprises the following steps:
(1) Cleaning stems and leaves of the lespedeza, drying, crushing and screening, and collecting undersize products to obtain lespedeza powder;
(2) Adding water into the lespedeza powder obtained in the step (1), stirring and dissolving, and crushing by using a high-pressure homogenizer to obtain a crushing liquid;
(3) Extracting the crushed liquid obtained in the step (2) with water, and filtering to obtain filtrate;
(4) Concentrating the filtrate obtained in the step (3) to be sticky, re-dissolving the filtrate by using ethanol, and filtering to obtain ethanol filtrate;
(5) Performing rotary evaporation on the ethanol filtrate obtained in the step (4), and performing freeze drying to obtain lespedeza extract powder;
(6) Dissolving the lespedeza extract powder obtained in the step (5) by using a propylene glycol-water solution, and filtering to obtain filtrate, namely the lespedeza extract.
The preparation method of the lespedeza extract specifically comprises the following steps:
(1) Pretreatment of lespedeza: cleaning stem and leaf of Lespedeza, naturally air-drying in shade and ventilation, pulverizing completely dried Lespedeza, sieving with 20-50 mesh sieve, and sealing and storing.
(2) Homogenizing under high pressure: preparing the prepared lespedeza powder into 2wt% aqueous solution, stirring and dissolving the lespedeza powder, and crushing the solution by a high-pressure homogenizer under the treatment condition of 200-300Bar for three times to obtain crushing solution.
(3) Water extraction: and (3) extracting the crushed liquid after high-pressure homogenization with water for 4 hours at the temperature of 45+/-5 ℃, synchronously carrying out ultrasonic treatment in the water extraction process, carrying out suction filtration after the water extraction is finished to obtain filtrate, and sealing and preserving.
(4) Spin distillation and concentration: concentrating the filtrate to be sticky, re-dissolving with 3-4 times of 95% ethanol, refrigerating at 4deg.C for 24 hr, and vacuum filtering to obtain ethanol filtrate.
(5) Spin-steaming and freeze-drying: carrying out rotary steaming on the ethanol filtrate, wherein the rotary steaming temperature is 50+/-5 ℃, the rotary steaming is carried out until the ethanol filtrate is sticky, putting the sample into a glass beaker, freezing the sample in a refrigerator at the temperature of minus 20 ℃ for 24 hours, and then carrying out freeze drying on the frozen sample in a vacuum freeze drying box, wherein the freeze drying working conditions are as follows: the temperature of the cold hydrazine is-60+/-5 ℃ and the pressure is 1-10Pa, and the lespedeza extract powder is obtained after the drying is finished.
(6) Preparing a solution: dissolving the obtained Lespedeza extract powder with a certain amount of propylene glycol-water solution, and filtering to obtain filtrate as Lespedeza extract.
Among the above-mentioned modulators, the biorhythms are preferably CLOCK gene and Per1 gene.
As a general technical conception, the invention also provides application of the regulator for the biological rhythm gene expression level in skin care products.
As a general technical concept, the invention also provides a skin care product, which comprises the regulator of the expression level of the biological rhythm genes.
As a general technical concept, the invention also provides application of the regulator of the biological rhythm gene expression level in cosmetics.
The present invention also provides, as a general technical idea, a cosmetic comprising the above regulator of the expression level of biorhythmic genes.
The skin care product and other components in the cosmetic can be used without limitation, and conventional components in the prior art can be used, for example, the other components of the skin care product can be found in CN110448478A.
The main components of the regulator comprise a lespedeza extract, a skin conditioner A containing glycoprotein and amino acid, a skin conditioner B containing glutamylaminoethyl imidazole and adenosine, and the relevant properties of the components are as follows:
lespedeza (Latin chemical name: lespedeza bicolor Turcz) is also known as: securinega suffruticosa, hu Zhitiao, sapium, pallet et al, belonging to Rosales, lespedeza genus of Lespedeza family of Leguminosae, with multiple branches, ovum-like leaf, and red-purple corolla. The oblique oval shape of the pods is distributed over about 60 species worldwide, from east asia to north eastern australia and north america. The plant belongs to a plurality of plants used as folk herbal medicines, has the effects of clearing heat and detoxicating, moistening lung and relieving cough, inducing diuresis and detumescence, promoting blood circulation and relieving pain, and is mainly used for treating common cold and fever, cough due to lung heat, traumatic injury, rheumatalgia, stranguria and the like. The Lespedeza plants mainly contain chemical components such as flavone, terpenes, alkaloids, sterols, organic acids, tannins, etc., and have pharmacological activities such as anti-inflammatory, antiallergic, antipruritic, analgesic, etc.
Adenosine is a nucleoside composed of N-9 of adenine and C-1 of D-ribose via beta-N9-glycosidic bond, and has the chemical formula of C 10 H 13 N 5 O 4 The phosphate is adenylate. Adenosine is an endogenous nucleoside distributed over human cells, and can directly enter cardiac muscle to generate adenylate through phosphorylation, participate in cardiac muscle energy metabolism, and simultaneously participate in dilating coronary vessels and increasing blood flow. Adenosine has physiological effects on the cardiovascular system and many other systems and tissues of the body. Adenosine is an important intermediate for the synthesis of Adenosine Triphosphate (ATP), adenine, adenylate, arabinoside. The molecular structural formula of adenosine is shown below:
adenosine not only can provide abundant nutrition for cells, but also can promote the synthesis of cell energy and improve the regeneration metabolic capacity of cells. Adenosine stimulates collagen production in dermis, accelerates DNA synthesis, counteracts skin aging process, and thus improves skin appearance. Jean-baptite Galey et al found that adenosine and its analogs can relax skin-contracting cells, meet the requirements of facial skin relaxation, smooth or lighten expression lines, relax facial features, reduce wrinkles, smiles, etc.
The skin conditioner A mainly comprises yeast glycoprotein, glutamic acid, valine, threonine, preservative, stabilizer and water. The skin conditioner A is preferably beer yeast as a strain, is fermented by biotechnology under aerobic condition, is essence extracted from beer yeast, is rich in glycoprotein and amino acid, has the effects of accelerating epidermal cell renewal, ensuring ATP to be at high level throughout the day, promoting glycolysis and mitochondrial respiration when the skin is in need, and can restore skin health by increasing cell energy level. In addition, the skin-care agent can stimulate key genes, improve the function of mitochondria under ultraviolet pressure, further realize youthful and light-refreshing complexion, improve the expression level of low circadian rhythms of skin, and be helpful for the skin to be in a full state throughout the day.
Skin conditioner B comprises glutamyl aminoethyl imidazole of formula C and preservative and water 10 H 16 N 4 O 3 Is a compound formed by amide condensation of terminal amino groups of glutamic acid with alpha-carboxyl group on histamine. Glutaminyl ethylimidazole, an anti-aging biorhythmic synchronizing peptide, is used to promote expression of the natural periodic circadian genes of the skin, thereby achieving an optimal correlation between the physiological activity of skin cells and environmental constraints. It has the following effects: optimizing natural defenses of keratinocytes and fibroblasts, promoting expression of vitamin D bioconversion enzymes, improving turnover and regeneration capacity of keratinocytes and keratinocytes, and promoting skin microcirculation by detoxification and nutrient supply. In recent years, due to the above-mentioned effects of glutaminyl ethyl imidazole, it is widely used in cosmetics as a raw material having an anti-aging effect, and the demand is expanding year by year. The molecular structural formula of glutamyl amino ethyl imidazole is shown as follows:
glutaminoethylimidazole is capable of mimicking the activation of circadian genes by sunlight. Studies on the human re-established epidermis have shown that circadian genes such as CLOCK and Per1 are cyclically activated in their biological rhythms with UV irradiation. The human re-established epidermis was placed in a dark environment and stimulated with glutamyl aminoethyl imidazole, which showed the ability to mimic UV signals and induce expression of circadian genes.
The accelerator of the present invention comprises a skin conditioner a consisting essentially of glycoprotein, amino acid, a skin conditioner B containing glutamyl aminoethyl imidazole, and adenosine, and the main efficacy of the accelerator can be seen in CN110448478A. Further, the research shows that the promoter has a certain promoting effect on the expression of biological rhythm genes, but has limited promoting effect, especially has a general promoting effect on the expression of CLOCK genes and Per1 genes. The CLOCK gene and the Per1 gene are important biological rhythm genes, promote the expression of the CLOCK gene and the Per1 gene, and are favorable for the efficient exertion of the actions of the biological rhythm genes. The research shows that by utilizing the synergistic effect of the accelerator and the lespedeza extract, the action of the lespedeza extract on the accelerator is obvious, the expression of the CLOCK gene and the Per1 gene by the accelerator can be efficiently promoted, the skin problem can be solved endogenously, the damage of skin DNA can be reduced, and the repair of cell scratch damage can be promoted. Meanwhile, the research shows that the synergistic effect of the accelerator and the lespedeza extract and the dosage proportion of the accelerator and the lespedeza extract have obvious influence, and excessive or insufficient dosage of the lespedeza can influence the acceleration effect of the accelerator on the expression of the CLOCK gene and the Per1 gene, and the more preferable dosage proportion is 1 time of the quality of the accelerator.
Moreover, the lespedeza extract adopted by the invention also has the effect of promoting the expression of Bmal1, per2 and Cry genes, can form complementation with the CLOCK gene and the Per1 gene, and can act on a rhythm gene loop in an omnibearing manner to realize superposition synergy, thereby restoring unbalanced biological rhythms to a normal healthy state, solving the problem of skin endogenously, remarkably reducing skin DNA damage and reducing skin blue light damage and photoaging.
The three raw materials in the accelerator have higher biocompatibility, have higher affinity to organisms, and bring more ideal safety coefficient for products. The four raw materials in the invention are all easily water-soluble raw materials, so that the problem that the oil-soluble raw materials need to be added with an emulsifying agent is avoided. The combination of the invention also has a very wide range of applications for storage conditions, environment and pH, with a wide range of application scenarios, enabling its adaptation and application in most forms and systems of skin care products and cosmetics.
Compared with the prior art, the invention has the advantages that:
1. the regulator of the biological rhythm gene expression level comprises the accelerator and the lespedeza extract enhancer, and can efficiently promote the expression of the accelerator on the CLOCK gene and the Per1 gene by the interaction of the lespedeza extract and the accelerator, thereby reducing the damage of skin DNA and promoting the repair of cell scratch damage.
2. The regulator of the biological rhythm gene expression level of the invention comprises the accelerator and the enhancer which have extremely high biocompatibility, do not generate extra stimulation to skin, have high safety coefficient and wide application scene, have wide application space and can be widely applied to the fields of skin care products, cosmetics and the like.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a statistical plot of the relative increase in the amount of expression of the CLOCK gene for the regulator samples of examples 1-5 and comparative examples 1-5.
FIG. 2 is a statistical graph showing the relative increase rate of the expression level of Per1 gene in the regulator samples of examples 1 to 5 and comparative examples 1 to 5.
FIG. 3 is a statistical plot of the relative protection of cellular DNA genes by the regulator samples of examples 1-5 and comparative examples 1-5.
FIG. 4 is a statistical plot of the healing rate of cell scratches for the modulator samples of examples 1-5 and comparative examples 1-5.
Detailed Description
The present invention will be described more fully hereinafter with reference to the accompanying drawings, in which preferred embodiments are shown, for the purpose of illustrating the invention, but the scope of the invention is not limited to the specific embodiments shown.
Unless defined otherwise, all technical and scientific terms used hereinafter have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the scope of the present invention.
Unless otherwise specifically indicated, the various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or may be prepared by existing methods.
The accelerators in the following examples and comparative examples are composite materials independently developed by my department, and consist of glutamyl amino ethyl imidazole, glycoprotein, amino acid, adenosine and the like, and specific components and preparation methods are shown in CN110448478A. The research shows that the lespedeza extract enhancer has an accelerating effect on the composition mentioned in CN110448478A, and can promote the expression of the composition on the CLOCK gene and the Per1 gene. In order to demonstrate the synergistic effect of the accelerators emphasized by the present invention and the enhancer of the extract of Lespedeza, the following examples and comparative examples are mainly exemplified by the following accelerators in weight proportions. Specifically, the accelerators used in the following examples and comparative examples mainly comprise the following components in mass fraction:
skin conditioner a0.2g: comprises yeast glycoprotein (3%), glutamic acid (3%), valine (0.55%), threonine (0.55%), preservative (1.1%), stabilizer (0.05%) and the balance of water;
skin conditioner B0.2 g: comprises glutamyl amino ethyl imidazole (1%), preservative (0.4%) and water in balance;
adenosine 0.1g.
Specific components of the above-mentioned preservatives and stabilizers etc. can be found in CN110448478A.
The lespedeza extract in the following examples and comparative examples refers to a solution of the lespedeza extract powder dissolved in a propylene glycol-water solution, wherein the mass content of the lespedeza extract powder is 0.1-10% and the mass content of the propylene glycol is 30-50%.
The preparation method of the lespedeza extract specifically comprises the following steps:
(1) Pretreatment of lespedeza: cleaning stem and leaf of Lespedeza, naturally air-drying in shade and ventilation, pulverizing completely dried Lespedeza, sieving with 20-50 mesh sieve, and sealing and storing.
(2) Homogenizing under high pressure: preparing the prepared lespedeza powder into 2wt% aqueous solution, stirring and dissolving the lespedeza powder, and crushing the solution by a high-pressure homogenizer under the treatment condition of 200-300Bar for three times to obtain crushing solution.
(3) Water extraction: and (3) extracting the crushed liquid after high-pressure homogenization with water for 4 hours at the temperature of 45+/-5 ℃, synchronously carrying out ultrasonic treatment in the water extraction process, carrying out suction filtration after the water extraction is finished to obtain filtrate, and sealing and preserving.
(4) Spin distillation and concentration: concentrating the filtrate to be sticky, re-dissolving with 3-4 times of 95% ethanol, refrigerating at 4deg.C for 24 hr, and vacuum filtering to obtain ethanol filtrate.
(5) Spin-steaming and freeze-drying: carrying out rotary steaming on the ethanol filtrate, wherein the rotary steaming temperature is 50+/-5 ℃, the rotary steaming is carried out until the ethanol filtrate is sticky, putting the sample into a glass beaker, freezing the sample in a refrigerator at the temperature of minus 20 ℃ for 24 hours, and then carrying out freeze drying on the frozen sample in a vacuum freeze drying box, wherein the freeze drying working conditions are as follows: the temperature of the cold hydrazine is-60+/-5 ℃ and the pressure is 1-10Pa, and the lespedeza extract powder is obtained after the drying is finished.
(6) Dissolving the Lespedeza extract powder with propylene glycol-water solution, and filtering to obtain filtrate.
The lespedeza extract used in the following examples and comparative examples refers to a propylene glycol-water solution of lespedeza extract powder, wherein the mass content of the lespedeza extract powder is 1% and the mass content of propylene glycol is 50%.
The regulators of the biological rhythm gene expression amounts in the following examples and comparative examples were used by accurately weighing 0.1g of adenosine in a beaker, then accurately weighing 0.2g of skin conditioner A and 0.2g of skin conditioner B, adding a metered Lespedeza extract (by mass), then adding deionized water to a net content of 100g, and stirring uniformly to obtain a regulator sample.
Example 1:
a regulator of biological rhythm gene expression quantity, comprising an accelerator and an enhancer, wherein the accelerator comprises a skin conditioner A mainly composed of glycoprotein and amino acid, a skin conditioner B containing glutamyl amino ethyl imidazole and adenosine; the enhancer comprises Lespedeza extract. The mass ratio of the accelerator to the enhancer is shown in the following table 1.
Table 1: raw material ratio of the regulator in examples 1 to 5 and comparative examples 1 to 5
Examples 2 to 5:
the compositions of the regulators of the biological rhythm gene expression levels in examples 2 to 5 were the same as in example 1 except that the proportions of the accelerator and the enhancer were different, specifically referring to the above-mentioned Table 1.
Comparative example 1:
the regulator of the biological rhythm gene expression level of this comparative example includes only the promoter, specifically, see the above table 1.
Comparative examples 2 to 4:
the regulator of the biological rhythm gene expression level in comparative examples 2 to 4 includes only the enhancer, specifically, see the above table 1.
Comparative example 5:
the composition of the regulator of the biological rhythm gene expression level in comparative example 5 was the same as in example 1 except that the ratio of the promoter to the enhancer was different, specifically referring to the above-mentioned table 1.
The following verification tests were carried out on the properties of the regulator samples obtained in examples 1 to 5 and comparative examples 1 to 5, in particular as follows:
(1) CLOCK gene immunofluorescence assay
1) Test background:
and simulating the effect of the regulator sample on the CLOCK gene by reducing the expression level of the CLOCK gene after the human body is irradiated by blue light and then using the regulator sample to restore the expression of the gene.
2) The testing method comprises the following steps:
A. cell inoculation: the density of the fibroblast (HFF-1) suspension was adjusted to 1.5X10 4 Inoculating into 96-well plate at 37deg.C under 5% CO at 100 ul/well 2 Culturing in an incubator for 24 hours.
B. Experimental grouping: comparative examples and examples.
C. Blue light irradiation: the modeling was performed using Blue Light (Blue Light), specifically stated as adjusting the intensity of Blue Light to 90 irradiation, and irradiation was performed for 45min at a height of 3cm from the 96 well plate, to obtain a cell model after Blue Light irradiation.
D. Raw material efficacy detection: a sample of the regulator to be tested was added to the blue light irradiated cell model according to Table 1, 100 ul/well, placed at 37℃and 5% CO 2 And (5) standing and repairing for 24 hours in the incubator.
E. Cell immunofluorescent staining: obtaining fluorescent photos of different samples through sample collection, cell fixation, permeation, primary washing, sealing, primary antibody, secondary antibody and secondary washing and picture collection.
F. Image processing: the photographed fluorescence photographs were analyzed using ImageJ average fluorescence intensity.
G. Statistical analysis: data statistical analysis is carried out by using GraphPad Prism 8.0 software, a chart is drawn, metering data is expressed by x+/-s, inter-group differences are analyzed by using one-wayANOVA, and P < 0.05 is used as the difference, so that the statistical significance is achieved.
3) Test results:
the calculation formula of the relative growth rate of the CLOCK gene expression quantity comprises the following steps: c% = (C x -C 0 )/C 0 *100％；
Wherein C represents the relative increase rate of the CLOCK gene expression level;
C x expressing the gene expression value of the sample group after the cell test;
C 0 the positive group (Blue Light,45 min) gene expression values after the cell test are shown.
The data were collated to obtain the data shown in table 2 and fig. 1 below.
Table 2: relative increase rate of CLOCK Gene expression
From fig. 1 and table 2, it can be seen that the regulator provided by the invention improves the promotion effect on the CLOCK gene to a certain extent, compared with the positive group irradiated by blue light, the expression level of the CLOCK gene in cells is obviously improved after the regulator is used, and the synergistic effect between the components of the regulator provided by the invention can be obtained by comparing the change rates of the CLOCK gene between comparative examples 1-5 and examples 1-5, and the efficacy of the regulator is obviously reduced when part of the components are absent or the component ratio is not in accordance with the requirement.
(2) Per1 Gene immunofluorescence assay
1) Test background:
the effect of the regulator sample on the Per1 gene was analyzed by simulating the decrease in the expression level of the Per1 gene in humans after exposure to blue light and restoring the gene expression using the regulator sample.
2) The testing method comprises the following steps:
A. cell inoculation: the density of the fibroblast (HFF-1) suspension was adjusted to 1.5X10 4 Inoculating into 96-well plate at 37deg.C under 5% CO at 100 ul/well 2 Culturing in an incubator for 24 hours.
B. Experimental grouping: comparative examples and examples.
C. Blue light irradiation: the modeling was performed using Blue Light (Blue Light), specifically stated as adjusting the intensity of Blue Light to 90 irradiation, and irradiation was performed for 45min at a height of 3cm from the 96 well plate, to obtain a cell model after Blue Light irradiation.
D. Raw material efficacy detection: a sample of the regulator to be tested was added to the blue light irradiated cell model according to Table 1, 100 ul/well, placed at 37℃and 5% CO 2 And (5) standing and repairing for 24 hours in the incubator.
E. Cell immunofluorescent staining: collecting samples, fixing cells, washing for the first time, sealing, washing for the first time, washing for the second time, and collecting pictures to obtain fluorescent photos of different samples.
F. Image processing: the photographed fluorescence photographs were analyzed using ImageJ average fluorescence intensity.
G. Statistical analysis: data statistical analysis is carried out by using GraphPad Prism 8.0 software, a chart is drawn, metering data is expressed by x+/-s, inter-group differences are analyzed by using one-wayANOVA, and P < 0.05 is used as the difference, so that the statistical significance is achieved.
3) Test results:
calculation formula of Per1 Gene expression relative growth Rate: p% = (P x -P 0 )/P 0 *100％；
Wherein P represents the relative increase rate of the Per1 gene expression amount;
P x expressing the gene expression value of the sample group after the cell test;
P 0 the positive group (Blue Light,45 min) gene expression values after the cell test are shown.
The data were collated to obtain the data shown in table 3 and fig. 2 below.
Table 3: per1 Gene expression relative growth Rate
From fig. 2 and table 3, it can be seen that the regulator provided by the present invention improves the promotion effect on the Per1 gene to a certain extent, compared with the positive group irradiated with blue light, the expression level of the cell Per1 gene is significantly improved after the use of the regulator, and by comparing the rate of change of the Per1 gene between comparative examples 1 to 5 and examples 1 to 5, the synergistic effect between the components of the regulator of the present invention is obtained, and when some components are absent or the component ratio does not meet the requirements, the effect of the regulator is significantly reduced.
(3) DNA damage repair test
1) Test background:
and simulating the repair condition of DNA damaged cells of a human body after the human body is irradiated by UV, and analyzing the repair performance of DNA damage of the regulator sample by applying the regulator sample.
2) The testing method comprises the following steps:
A. cell inoculation: the density of the fibroblast (HFF-1) suspension was adjusted to 2X 10 4 Inoculating into 96-well plate at 37deg.C under 5% CO at 100 ul/well 2 Culturing in an incubator for 24 hours.
B. Experimental grouping: comparative examples and examples.
C. Model construction: removing liquid from positive control group by using UV stress mould, adding 50 μl PBS/well, and adjusting UV intensity to 100J/cm 2 Irradiation was performed for 10min at a height of 3cm from the 96-well plate.
D. Raw material efficacy detection: the samples of the modulators to be tested were added to the UV-irradiated cell model according to Table 1, 100 ul/well, placed at 37℃and 5% CO 2 And (5) standing and repairing for 24 hours in the incubator.
E. Cell immunofluorescent staining: and finally obtaining a photograph after fluorescent staining through primary washing, fixing, secondary washing, adding an antibody, adding a detection solution and performing fluorescent photographing.
F. Image processing: the photographed fluorescence photographs were analyzed using ImageJ average fluorescence intensity.
G. Statistical analysis: data statistical analysis is carried out by using GraphPad Prism 8.0 software, a chart is drawn, metering data is expressed by x+/-s, inter-group differences are analyzed by using one-wayANOVA, and P < 0.05 is used as the difference, so that the statistical significance is achieved.
3) Test results:
the calculation formula of the relative protection rate of the cell DNA gene: d% = (D 0 -D x )/D 0 *100％；
Wherein D represents the relative protection rate of the cellular DNA gene;
D x representing the relative fluorescence intensity of cell DNA of the sample group after the cell test;
D 0 the relative fluorescence intensity of the cell DNA of the positive group (UV, 10 min) after the cell test is shown.
The data were collated to obtain the data shown in table 4 and fig. 3 below.
Table 4: relative protection rate of cellular DNA genes
As can be seen from fig. 3 and table 4, the regulator provided by the present invention improves the protection and repair effects of the cell DNA after UV irradiation to a certain extent, and compared with the positive group using UV irradiation, the protection of the cell DNA after using the regulator is significantly improved, and by comparing the relative protection rates of the DNA between comparative examples 1 to 5 and examples 1 to 5, the synergistic effect of each component of the regulator provided by the present invention is shown, and when some components are absent or the component ratio is not satisfactory, the efficacy effect of the regulator is significantly reduced.
(4) Cell scratch test
1) Test principle:
simulating the migration speed of cells in vivo, marking out scratches on the growth plane of the cells to cause cell damage, and observing the effect of the sample on promoting cell healing after the regulator sample is added to analyze the healing efficacy of the regulator sample.
2) The test steps are as follows:
A. keratinocytes were resuscitated and cells were cultured with DMEM containing 15% fbs as a culture medium.
B. The ibidi scratch pad was placed in a 6-well plate. Taking log phase cells, digesting the cells with 0.25% pancreatin, and using MEM containing 15% FBS as culture medium, wherein the number of cells per well is 4.0X10 4 Inoculating 70 ul/well into ibidi mold, placing at 37deg.C, 5% CO 2 Culturing in an incubator for 24 hours.
C. The ibidi scratch insert was pulled out and samples were added, 2 mL/well, and added to the wells as in the comparative example and example in table 1. Placed at 37 ℃ and 5% CO 2 After 24h incubation in the incubator, the cell morphology was observed under a microscope and photographed.
D. And (3) calculating: the cell scratch healing rate was calculated as follows:
healing rate = (0 h scratch area-24 h scratch area after loading)/0 h scratch area 100%.
E. Statistical analysis: data statistical analysis is carried out by using GraphPad Prism 8.0 software, a chart is drawn, metering data is expressed by x+/-s, inter-group differences are analyzed by using one-wayANOVA, and P < 0.05 is used as the difference, so that the statistical significance is achieved.
3) Test results:
cell scratch healing rate: f% = (F 0 -F x )/F 0 *100％；
Wherein F represents the cell scratch healing rate;
F x representing the scratch area of the sample group for 24 hours after the cell test;
F 0 the scratch area of the negative group (PBS) 24h after the cell test is shown.
The data were collated to obtain the data shown in table 5 and fig. 4 below.
Table 5: cell scratch healing rate
As can be seen from fig. 4 and table 5, the regulator provided by the present invention improves the repair rate of cell scratches to a certain extent, and compared with the negative group, the repair rate of cell scratches after using the regulator is significantly improved, and by comparing the repair rates of cell scratches between comparative examples 1 to 5 and examples 1 to 5, the synergistic effect between the components of the regulator provided by the present invention is known, and when some components are absent or the component ratio is not satisfactory, the efficacy of the regulator is significantly reduced.
Example 6:
the regulator of the biological rhythm gene expression level in example 3 is used for preparing skin care products and can comprise a regulator, a humectant, a penetration enhancer, a cutin softener and a preservative, wherein the mass content of each component can be 1%, 9%, 1%, 0.4% and 1% respectively, and the balance is water, the humectant can be glycerin and 1,3 butanediol, the penetration enhancer can be pentanediol, the cutin softener can be hydroxyethylpiperazine ethane sulfonic acid, and the preservative can be PHL. The preparation process of the skin care product in this example can be seen in CN110448478A.
The regulator of biorhythmic gene expression level in example 3 was also used for preparing cosmetics, and about 1% of the regulator in example 3 was added to conventional cosmetics.
Claims (10)
1. A regulator of biological rhythm gene expression quantity, comprising a promoter, wherein the promoter comprises a skin conditioner A mainly composed of glycoprotein and amino acid, a skin conditioner B containing glutamylaminoethyl imidazole and adenosine; the regulator is characterized by further comprising an enhancer, wherein the enhancer comprises a lespedeza extract.
2. The regulator according to claim 1, wherein the amount of the lespedeza extract is 0.02 to 10 times by mass of the accelerator.
3. The regulator according to claim 2, wherein the amount of the lespedeza extract is 0.2 to 6 times the mass of the accelerator.
4. A regulator according to any one of claims 1 to 3, wherein the lespedeza extract is a solution of lespedeza extract powder dissolved in a propylene glycol-water solution, wherein the mass content of the lespedeza extract powder is 0.1 to 10% and the mass content of propylene glycol is 30 to 50%.
5. The regulator according to claim 4, wherein the preparation method of the lespedeza extract comprises the following steps:
(1) Cleaning stems and leaves of the lespedeza, drying, crushing and screening, and collecting undersize products to obtain lespedeza powder;
(2) Adding water into the lespedeza powder obtained in the step (1), stirring and dissolving, and crushing by using a high-pressure homogenizer to obtain a crushing liquid;
(3) Extracting the crushed liquid obtained in the step (2) with water, and filtering to obtain filtrate;
(4) Concentrating the filtrate obtained in the step (3) to be sticky, re-dissolving the filtrate by using ethanol, and filtering to obtain ethanol filtrate;
(5) Performing rotary evaporation on the ethanol filtrate obtained in the step (4), and performing freeze drying to obtain lespedeza extract powder;
(6) Dissolving the lespedeza extract powder obtained in the step (5) by using a propylene glycol-water solution, and filtering to obtain filtrate, namely the lespedeza extract.
6. A modulator according to any one of claims 1 to 3 wherein the biorhythms are CLOCK and Per1 genes.
7. Use of the regulator of biorhythmic gene expression level according to any one of claims 1 to 6 in skin care products.
8. A skin care product comprising the regulator for the expression level of biorhythmic genes according to any one of claims 1 to 6.
9. Use of the regulator of biorhythmic gene expression level according to any one of claims 1 to 6 in cosmetics.
10. A cosmetic comprising the regulator of the expression level of a biorhythmic gene according to any one of claims 1 to 6.
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Cited By (1)
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| CN117942276A (en) * | 2024-03-27 | 2024-04-30 | 南京斯拜科生物科技股份有限公司 | Compound peptide for realizing percutaneous absorption promotion of fibroblast proliferation under low concentration and preparation method thereof |
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| CN109481492A (en) * | 2017-09-13 | 2019-03-19 | 韩国百鸥思特公司 | For preventing or improving the composition of circadian disorders |
| CN109939058A (en) * | 2019-04-19 | 2019-06-28 | 广州萝薇化妆品有限公司 | A kind of cosmetic composition of anti-blue light and preparation method thereof |
| CN110448478A (en) * | 2019-09-26 | 2019-11-15 | 湖南御家化妆品制造有限公司 | Composition and application thereof in preparing skin care product for regulating biological rhythm of skin |
| KR20200132261A (en) * | 2019-05-16 | 2020-11-25 | 경희대학교 산학협력단 | Composition for prevention and treatment of muscle atrophy comprising lespedeza bicolor extract |
| CN112870124A (en) * | 2021-02-02 | 2021-06-01 | 广东梵蜜琳生物科技有限公司 | Composition for regulating time rhythm and preparation method and application thereof |
| US20210169960A1 (en) * | 2018-07-27 | 2021-06-10 | Pierre Fabre Dermo-Cosmetique | Lespedeza capitata extract for use in the field of hair care |
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| CN109481492A (en) * | 2017-09-13 | 2019-03-19 | 韩国百鸥思特公司 | For preventing or improving the composition of circadian disorders |
| US20210169960A1 (en) * | 2018-07-27 | 2021-06-10 | Pierre Fabre Dermo-Cosmetique | Lespedeza capitata extract for use in the field of hair care |
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| CN117942276A (en) * | 2024-03-27 | 2024-04-30 | 南京斯拜科生物科技股份有限公司 | Compound peptide for realizing percutaneous absorption promotion of fibroblast proliferation under low concentration and preparation method thereof |
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