CN117959226A - Photodamage-resistant rice fermentation liquor mixture freeze-dried powder and preparation method and application thereof - Google Patents
Photodamage-resistant rice fermentation liquor mixture freeze-dried powder and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to photodamage-resistant rice fermentation liquor mixture freeze-dried powder, and a preparation method and application thereof. Dissolving a mixture consisting of rice fermentation liquor and sturgeon collagen and mannitol in water for injection according to the mixture and mannitol, adding active carbon, uniformly stirring, filtering by using a filter cloth and a microporous sterilization filter, filling the mixture into a sterilized container, and transferring the container into a freeze dryer under the aseptic condition; and then performing prefreezing, decompressing, sublimating and drying to obtain the photodamage-resistant rice fermentation broth mixture freeze-dried powder. The obtained freeze-dried product has low moisture content, good stability, good appearance and good re-solubility. Compared with the prior art, the photodamage-resistant rice fermentation liquor mixture freeze-dried powder provided by the invention contains abundant succinic acid, is favorable for promoting cell renewal, controlling sebum production, effectively resisting inflammation and bacteria, and is milder and has no stimulation compared with other maintenance acids.
Description
Technical Field
The invention belongs to the technical field of daily chemicals, and particularly relates to photodamage-resistant rice fermentation liquor mixture freeze-dried powder and a preparation method and application thereof.
Background
The rice fermentation liquor is a natural rice bran fermentation product, is rich in various effective components such as gamma-oryzanol, biological polysaccharide, amino acid and the like, can activate glutathione reductase activity, inhibit the activity of the neuraminidase, has the effects of whitening skin, inhibiting melanin growth and the like, and can promote the generation of cholesterol, cerebral amide and sebum, improve the softness of skin, and have the effects of resisting oxidation, conditioning, activating skin, resisting aging and the like.
Collagen and elastin together maintain the firmness and flexibility of the skin, collagen plumps the skin, and elastin makes the skin flexible. However, with age, the levels of both collagen and elastin decrease, resulting in wrinkling and sagging of the skin. In recent years, some collagens extracted from fish and fish processing byproducts have been more focused and studied, such as the collagen of tilapia skin, the collagen of large-eye tuna skin, and sturgeon collagen, which have been reported.
The lyophilization method is to freeze the water in the product to be dried at low temperature, then dry the product in vacuum environment to sublimate the water from solid state into water vapor and eliminate the water from the product to dry the product actively. The method effectively protects the stability of the active ingredients of a plurality of thermosensitive biological products; secondly, the freeze-dried product is fluffy after being dried, and can be quickly dissolved and restore the physical and chemical properties and biological activity of the original aqueous solution after being added with water; third, the vacuum condition has good protection effect on the easily oxidized substances.
With the increasing serious pollution of the atmosphere, the destruction of the ozone layer causes the increase of the surface ultraviolet radiation, and various adverse effects are generated on the human body. The long-wave Ultraviolet (UVA) in the natural world accounts for about 97 percent, UVB accounts for only 3 percent, and researches show that the penetrating power of UVA to skin is far higher than that of UVB, and the UVA can penetrate into the dermis layer to cause damage of collagen and elastic fiber in the dermis layer so as to further cause oxidative damage in a deeper layer, and light-related skin diseases such as photoaging, various skin cancers, photosensitive reactions, phototoxic reactions and the like are generated.
In the prior art, preventive and/or therapeutic agents for skin damage caused by ultraviolet rays include: an ultraviolet scattering agent (such as titanium oxide) for blocking skin absorption of ultraviolet rays, an ultraviolet absorbing agent (such as p-methoxy cinnamic acid-2-ethylhexyl), or an antioxidant for removing free radicals generated by ultraviolet rays. However, ultraviolet scattering agents and ultraviolet absorbing agents are effective for outdoor sunscreening, but are not suitable for daily use. In addition, antioxidants have problems in terms of stability and safety.
Disclosure of Invention
Based on the problems of the preventive and/or therapeutic agents for skin injury caused by ultraviolet rays described in the background art, the invention provides a photodamage-resistant rice fermentation broth mixture freeze-dried powder, and a preparation method and application thereof.
Since the prior art does not relate to whether rice fermentation broth or its lyophilized powder can inhibit the chemokine CXCL 2. The invention provides photodamage-resistant rice fermentation liquor mixture freeze-dried powder and a preparation method and application thereof from the aspect of inhibiting the production of chemokine CXCL 2.
The aim of the invention can be achieved by the following technical scheme:
the invention firstly provides a preparation method of photodamage-resistant rice fermentation liquor mixture freeze-dried powder, which comprises the following steps:
(1) Dissolving a mixture consisting of rice fermentation liquor and sturgeon collagen and mannitol in water for injection, adding active carbon, uniformly stirring, and filtering out a primary filtrate by using filter cloth;
(2) Filtering the primary filtrate obtained in the step (1) by using a microporous sterilization filter to obtain secondary filtrate, canning the secondary filtrate into a sterilized container, and transferring the container into a freeze dryer under the aseptic condition;
(3) Primarily freezing the container filled with the secondary filtrate at the temperature of-20 to-10 ℃; further cooling to-50 to-40 ℃ (for example, -45 ℃), and freezing for 2-3 hours to completely freeze the mixture; then the freezing temperature is stably increased to-25 to-15 ℃, vacuumizing is carried out, the temperature is increased to-5 to 0 ℃ within 10 to 12 hours, and the temperature is maintained for 4 to 8 hours (for example, 6 hours); and heating to 35-38 ℃ within 2-3 hours, maintaining the temperature and extreme vacuum to carry out secondary analysis and dehydration for 3-4 hours, and exhausting after dehydration is finished to obtain the photodamage-resistant rice fermentation liquor mixture freeze-dried powder.
In one embodiment of the invention, in step (1), the mixture consisting of rice fermentation broth and sturgeon collagen is mixed with mannitol in a ratio of 1-3:1 by volume of the mixture to mannitol, preferably in a ratio of 3:2, the volume ratio of the sum of the volumes of the mixture and mannitol to the volume ratio of the water for injection is 1:2-4.
In one embodiment of the invention, in the step (1), the mass ratio of the rice fermentation liquor to the sturgeon collagen in the mixture consisting of the rice fermentation liquor and the sturgeon collagen is 1-4:1, preferably 2:1.
Sturgeon collagen can promote the generation of type I collagen in fibroblasts and activate TGF-beta; the rice fermentation liquor can reduce CXCL2 chemokine expression, lighten damage of UVA to fibroblast fibers, and the two can realize multi-channel antioxidation by compounding, thereby playing a role in synergistic anti-aging.
In the invention, mannitol in the step (1) is an excipient, and has good fluffy framework and redissolution effect; the activated carbon is an adsorbent, is used for adsorbing redundant solvent, and has no special requirement on the addition amount of the activated carbon.
In one embodiment of the present invention, in the step (1), the mesh number of the filter cloth is 800 mesh to 5000 mesh (corresponding to a pore size of 15 μm to 2.6 μm), preferably 1000 mesh filter cloth (corresponding to a pore size of 13 μm) is used.
In one embodiment of the present invention, in the step (2), the pore size of the microporous sterilization filter is 0.22 to 0.3 μm (preferably 0.22 μm) for the purpose of isolating activated carbon and microorganisms, etc.
In the invention, in the step (3), the preparation process flow of the freeze-dried powder is primary freezing, decompression, sublimation and drying, the primary freezing temperature is generally 10-20 ℃ at the eutectic temperature, and the water content in the mixed solution of the rice fermentation liquid and mannitol is higher, so that the water is generally calculated according to the freezing point of water, namely 0 ℃, the temperature of minus 20-minus 10 ℃ is more suitable, and the primary freezing is carried out. And further cooling to-50 to-40 ℃ (for example, -45 ℃), so that the water is completely frozen, and bottle spraying is avoided. And then the material is sublimated by vacuumizing and heating, and the upper material is dried first during sublimation, so that if the temperature rises too fast, the collapse temperature can be reached, the moisture content of the final product is affected, and the temperature close to the eutectic point is selected. After sublimation drying, a part of adsorbed water remains, and the temperature needs to be raised again for removal. The obtained freeze-dried product has low moisture content, good stability, good appearance and good re-solubility.
The invention further provides photodamage-resistant rice fermentation liquor mixture freeze-dried powder obtained based on the preparation method. The photodamage-resistant rice fermentation liquor mixture freeze-dried powder has good stability, redissolution and appearance. The photodamage-resistant rice fermentation liquor mixture freeze-dried powder obtained by the invention is placed in a container and is stored in a sealed manner in a sterile environment.
The invention further provides application of the photodamage-resistant rice fermentation liquor mixture freeze-dried powder obtained based on the preparation method in cosmetics:
The photodamage-resistant rice fermentation liquor mixture freeze-dried powder can form related cosmetics with a cosmetically acceptable carrier or excipient, and the cosmetics can be daily chemical products such as facial cleanser, toning lotion, skin cleaning lotion, balancing water, emulsion, cream, facial mask, bath lotion and the like.
Furthermore, the invention also provides an essence emulsion containing photodamage-resistant rice fermentation liquor mixture freeze-dried powder, which comprises the following components
The rice fermentation liquor mixture freeze-dried powder is the photodamage-resistant rice fermentation liquor mixture freeze-dried powder prepared by the preparation method.
The invention further provides a preparation method of the essence emulsion containing the photodamage-resistant rice fermentation liquor mixture freeze-dried powder, which comprises the following steps:
(1) Mixing the components of the component A, stirring for 15-35 min at 20-50 ℃ and stirring speed of 150-300rpm to form an oil phase;
(2) Mixing the components of the component B, stirring for 10-25 min at 20-50 ℃ and the stirring speed is 300-500 rpm to form a water phase;
(3) Mixing and stirring the oil phase and the water phase at 20-50 ℃ for 5-20 min, adding essence, bromopol and propylparaben, stirring again at 20-50 ℃ for 10-30 min at 200-400 rpm, and passing through a nano homogenizer for 3-8 times to obtain the essence emulsion frame.
(4) When in use, the D phase is uniformly mixed with the essence emulsion frame.
The photodamage-resistant rice fermentation liquor mixture freeze-dried powder prepared by the invention has stable property, is safe and effective, is rich in various effective components, has rich succinic acid content, is favorable for promoting cell renewal, controlling sebum production, is effective in anti-inflammatory and antibacterial, and is milder and has no stimulation compared with other maintenance acids. The preparation method of the photodamage-resistant rice fermentation liquor mixture freeze-dried powder is simple, and the original biological activity is maintained while the property is stable.
Chemokine CXCL2 is a small cytokine or signal protein secreted by cells and is closely related to the production of inflammation, and inhibition of the production of chemokine CXCL2 can inhibit the production of pro-inflammatory cytokines and the aging apoptosis of keratinocytes, maintain the collagen homeostasis secreted by fibroblasts, and further inhibit aging. The photodamage-resistant rice fermentation liquor mixture freeze-dried powder provided by the invention can effectively reduce the expression quantity of chemotactic factor CXCL2, lighten the oxidative damage of UVA to human skin fibroblasts and has good anti-aging effect.
Compared with the prior art, the invention has the following advantages:
1. The photodamage-resistant rice fermentation liquor mixture freeze-dried powder provided by the invention contains abundant succinic acid, is favorable for promoting cell renewal, controlling sebum production, is effective in anti-inflammatory and antibacterial, and is milder and free of stimulation compared with other maintenance acids.
2. The preparation method of the photodamage-resistant rice fermentation liquor mixture freeze-dried powder is simple, maintains the original bioactivity and stable property, can effectively reduce the expression quantity of the chemotactic factor CXCL2, lightens the oxidative damage of UVA to human skin fibroblasts, and has good anti-aging effect. Photoaging damage is a complex process and has a series of influencing factors, and the theory and application research of chemokines are one of hot spots of current life research, and the research shows that the chemokine CXCL2 as p53 target genes mediates DNA damage to induce cell aging. According to the invention, photodamage-resistant rice fermentation liquor mixture freeze-dried powder composed of rice fermentation liquor and sturgeon collagen is studied, so that the expression quantity of chemotactic factor CXCL2 can be effectively reduced, the damage of UVA to fibroblast fibers is lightened, the generation of type I collagen in the fibroblast cells can be promoted, TGF-beta is activated, and the two components can be compounded to realize multi-channel antioxidation, thereby playing a role in synergistic anti-aging. The research of the invention discovers that photodamage-resistant rice fermentation liquor mixture freeze-dried powder consisting of rice fermentation liquor and sturgeon collagen can play an anti-aging role from a gene level.
Drawings
Fig. 1: the GO function enrichment analysis results of different differential genes;
fig. 2: differential gene KEGG pathway enrichment analysis results;
fig. 3: results with different chemokines affected in different groups.
Detailed Description
The invention will now be described in detail with reference to the drawings and specific examples.
It will be apparent to those skilled in the art that the examples are merely to aid in understanding the invention and are not to be construed as a specific limitation thereof. Other non-essential modifications and adaptations of the invention are within the scope of the invention. The following percentages (%) are mass percentages unless otherwise indicated.
Sturgeon collagen was purchased from Shanghai running script and Biotechnology Inc.
The preparation method of the rice fermentation liquor comprises the following steps:
(1) Adding water into rice powder, heating, stirring to obtain rice slurry, adding beta-glucanase into 60wt% of rice slurry, wherein the addition amount of enzyme is 0.5% of the mass of the rice slurry, and the enzyme activity is 150U/g, and performing enzymolysis to obtain rice slurry enzymatic hydrolysate;
(2) Inoculating yeast strain Saccharomyces veronae into the rice milk enzymolysis liquid, and fermenting under the following conditions: fermenting at 40deg.C for 48 hr with pH of 6-6.5 to obtain fermentation broth;
(3) Concentrating the fermentation liquor by 10 times through reverse osmosis technology, efficiently enriching active substances, and obtaining rice fermentation liquor, namely yeast/rice fermentation product filtrate containing the active substances through heating sterilization and cooling filtration.
Example 1
The preparation method of the photodamage-resistant rice fermentation liquor mixture freeze-dried powder comprises the following steps:
(1) Mixing rice fermentation liquor mixture (the mass ratio of rice fermentation liquor to sturgeon collagen in the mixture is 2:1) with mannitol solution according to the following ratio of 3:2 volume ratio of the rice fermentation liquid mixture to mannitol is dissolved in water for injection, and the volume ratio of the rice fermentation liquid mixture to the mannitol to the water for injection is 1:2, adding active carbon, uniformly stirring, and filtering out primary filtrate by using 1000-mesh filter cloth.
(2) Filtering the primary filtrate with a microporous sterilization filter of 0.22 μm to obtain secondary filtrate, canning the secondary filtrate into sterilized container, sealing the container under aseptic condition, and transferring into freeze dryer.
(3) Placing the filtrate into a freeze dryer at the temperature of minus 20 ℃ to minus 18 ℃ for preliminary freezing for 3 hours; further cooling to-45 ℃, and freezing for 2-3 hours to completely freeze the mixture; then the freezing stability is raised to about minus 25 ℃, vacuum pumping is carried out, the temperature is raised to minus 5 to 0 ℃ within 10 to 12 hours, and the temperature is maintained for 6 hours; and heating to 35 ℃ within 2-3 hours, maintaining the temperature to be in extreme vacuum for secondary analysis and dehydration for 3 hours, and exhausting after dehydration is finished to obtain the photodamage-resistant rice fermentation liquor mixture freeze-dried powder.
Example 2
The preparation method of the photodamage-resistant rice fermentation liquor mixture freeze-dried powder comprises the following steps:
(1) Mixing rice fermentation liquor mixture (the mass ratio of rice fermentation liquor to sturgeon collagen in the mixture is 3:1) with mannitol solution according to the following ratio of 1:1 volume ratio is dissolved in water for injection, and the volume ratio of the sum of the volumes of the rice fermentation liquid mixture and mannitol to the water for injection is 1:3, adding active carbon, uniformly stirring, and filtering out primary filtrate by using 1000-mesh filter cloth.
(2) Filtering the primary filtrate with a microporous sterilization filter of 0.22 μm to obtain secondary filtrate, canning the secondary filtrate into sterilized container, sealing the container under aseptic condition, and transferring into freeze dryer.
(3) Placing the filtrate into a freeze dryer at the temperature of minus 20 ℃ to minus 18 ℃ for preliminary freezing for 3 hours; further cooling to-45 ℃, and freezing for 2-3 hours to completely freeze the mixture; then the freezing stability is raised to about minus 25 ℃, vacuum pumping is carried out, the temperature is raised to minus 5 to 0 ℃ within 10 to 12 hours, and the temperature is maintained for 6 hours; and heating to 35 ℃ within 2-3 hours, maintaining the temperature to be in extreme vacuum for secondary analysis and dehydration for 3 hours, and exhausting after dehydration is finished to obtain the photodamage-resistant rice fermentation liquor mixture freeze-dried powder.
Example 3
The preparation method of the photodamage-resistant rice fermentation liquor mixture freeze-dried powder comprises the following steps:
(1) Mixing rice fermentation liquor mixture (the mass ratio of rice fermentation liquor to sturgeon collagen in the mixture is 4:1) with mannitol solution according to the following ratio of 2:1 volume ratio is dissolved in water for injection, and the volume ratio of the sum of the volumes of the rice fermentation liquid mixture and mannitol to the water for injection is 1:4, adding active carbon, stirring uniformly, and filtering out primary filtrate by using 1000-mesh filter cloth.
(2) Filtering the primary filtrate with a microporous sterilization filter of 0.22 μm to obtain secondary filtrate, canning the secondary filtrate into sterilized container, sealing the container under aseptic condition, and transferring into freeze dryer.
(3) Placing the filtrate into a freeze dryer at the temperature of minus 20 ℃ to minus 18 ℃ for preliminary freezing for 3 hours; further cooling to-45 ℃, and freezing for 2-3 hours to completely freeze the mixture; then the freezing stability is raised to about minus 25 ℃, vacuum pumping is carried out, the temperature is raised to minus 5 to 0 ℃ within 10 to 12 hours, and the temperature is maintained for 6 hours; and heating to 35 ℃ within 2-3 hours, maintaining the temperature to be in extreme vacuum for secondary analysis and dehydration for 3 hours, and exhausting after dehydration is finished to obtain the photodamage-resistant rice fermentation liquor mixture freeze-dried powder.
Comparative example 1
The preparation method of the photodamage-resistant rice fermentation liquor mixture freeze-dried powder comprises the following steps:
(1) Mixing rice fermentation liquor mixture (the mass ratio of rice fermentation liquor to tuna collagen in the mixture is 2:1) with mannitol solution according to the following ratio of 3:2 volume ratio of the rice fermentation liquid mixture to mannitol is dissolved in water for injection, and the volume ratio of the rice fermentation liquid mixture to the mannitol to the water for injection is 1:2, adding active carbon, uniformly stirring, and filtering out primary filtrate by using 1000-mesh filter cloth.
(2) Filtering the primary filtrate with a microporous sterilization filter of 0.22 μm to obtain secondary filtrate, canning the secondary filtrate into sterilized container, sealing the container under aseptic condition, and transferring into freeze dryer.
(3) Placing the filtrate into a freeze dryer at the temperature of minus 20 ℃ to minus 18 ℃ for preliminary freezing for 3 hours; further cooling to-45 ℃, and freezing for 2-3 hours to completely freeze the mixture; then the freezing stability is raised to about minus 25 ℃, vacuum pumping is carried out, the temperature is raised to minus 5 to 0 ℃ within 10 to 12 hours, and the temperature is maintained for 6 hours; and heating to 35 ℃ within 2-3 hours, maintaining the temperature to be in extreme vacuum for secondary analysis and dehydration for 3 hours, and exhausting after dehydration is finished to obtain the photodamage-resistant rice fermentation liquor mixture freeze-dried powder.
Example 4
Influence of photodamage-resistant Rice fermentation broth mixture lyophilized powder of example 1 and succinic acid on Long-wave Ultraviolet (UVA) injured human skin fibroblasts
(1) WST-8 method for detecting total SOD activity
(1.1) Inoculating human skin fibroblast HES into a cell culture dish according to proper cell density, and placing into a CO 2 incubator for incubation until the cell fusion rate is about 80%;
(1.2) adding the test substances to the cells according to the experimental groups for culturing for 24 hours, wherein the experimental groups are as follows:
blank control group: serum-free medium
Injury model group: serum-free medium
Sample group: the photodamage resistant rice fermentation broth mixture freeze-dried powder (1, 0.5, 0.25 mg/mL) and succinic acid (1, 0.5, 0.25 mg/mL) of example 1 were prepared with serum-free medium, respectively;
(1.3) cells of the injury model group and the sample group were subjected to UVA irradiation, except for the blank control group. The irradiation method comprises the following steps: 4 UVA parallel lamp tubes are adopted, after preheating for 30min, an ultraviolet irradiator is used for measuring the intensity, the distance between the culture plate and the lamp tubes is 5cm, and the UVA radiation dose is adjusted to be 2J/cm 2 by changing the radiation time.
(1.4) Adding a serum-free culture medium after the irradiation is finished, and continuously culturing for 3 hours;
(1.5) PBS was washed 1 time, and the SOD sample preparation solution provided in the Biyun Tian Total SOD Activity detection kit (WST-8 method) was added in a proportion of 100-200. Mu.l per 100 ten thousand cells, and properly blown to lyse the cells. Centrifuging at 4 ℃ for 3-5 minutes at about 12,000g, and taking supernatant as a sample to be detected;
(1.6) measuring protein concentration by using a Biyun Tian BCA protein concentration measuring kit, properly diluting a sample by using an SOD detection buffer according to the protein concentration and the predicted protein usage, and completing the subsequent measurement in the same day;
(1.7) preparing WST-8/enzyme working solution and reaction starting working solution: according to the configuration of the specification, the preparation is ready for use;
(1.8) sample measurement: sample holes and blank control holes are arranged by using a 96-well plate, a sample to be detected and other various solutions are added according to the proportion of the specification, the mixture is uniformly mixed (low-temperature operation) after the reaction starting working solution is added, the mixture is incubated at 37 ℃ for 30 minutes, and the absorbance is measured at 450 nm.
Superoxide dismutase (Superoxide Dismutase, SOD) is an important antioxidant enzyme in organisms.
The activity of SOD enzyme is calculated according to the formula,
Absorbance of control group, a ck, a E: absorbance of the test sample (sample set or injury model set), V: the total volume of the sample solution (which refers to the total volume of the mixed solution obtained by adding the sample to be measured and other various solutions according to the proportion of the specification in the step 1.8 and adding the reaction starting working solution) is V t: sample volume from sample fluid at test, W: fresh sample (refer to the weight of the mixed liquid after adding the sample to be tested and other various solutions according to the proportion of the instruction in the step 1.8 and adding the reaction starting working liquid).
The results are shown in Table 1, the SOD enzyme activity of the injury model group is reduced by 61.14% compared with that of the blank group, the P is less than 0.01, the difference has statistical significance, and the SOD enzyme activity can be improved after the mixture of the rice fermentation liquor with photodamage resistance and the freeze-dried powder of succinic acid are added for pretreatment for 24 hours.
TABLE 1 influence of lyophilized powder and succinic acid on SOD enzyme Activity of cells after UVA injury to fibroblasts
Note that: p <0.01 compared to blank group
(2) High throughput transcriptomic assay
(2.1) Inoculating human skin fibroblast HES into a cell culture dish according to proper cell density, and placing into a CO 2 incubator for incubation until the cell fusion rate is about 80%;
(2.2) adding the test substances to the cells according to the experimental groups for culturing for 24 hours, wherein the experimental groups are as follows:
blank control group: serum-free medium
Injury model group: serum-free medium
Sample group: preparing freeze-dried powder (1 mg/mL) of the photodamage-resistant rice fermentation broth mixture of the example 1 and succinic acid (1 mg/mL) respectively by using a serum-free culture medium;
(2.3) cells except for the blank control were subjected to UVA irradiation. The irradiation method comprises the following steps: 4 UVA parallel lamp tubes are adopted, after preheating for 30min, an ultraviolet irradiator is used for measuring the intensity, the distance between the culture plate and the lamp tubes is 5cm, and the UVA radiation dose is adjusted to be 2J/cm 2 by changing the radiation time.
(2.4) Adding a serum-free culture medium after the irradiation is finished, and continuously culturing for 3 hours;
(2.5) discarding the culture solution, washing 2 times with PBS, adding 1.5mL of Trizol solution, blowing the lysed cells thoroughly, collecting the cells and placing the cells in a 1.5mL EP tube, preserving the cells at-80 ℃, and carrying out sequencing.
MRNA transcriptome sequencing, namely sequencing analysis by using a high-throughput sequencing technology, has the advantages of high sensitivity and comprehensiveness, can analyze differential expression of genes, and plays an important role in elucidating biological development and disease occurrence and development. Setting 3 groups of blank control group samples K to reduce errors, wherein the marks are K1, K2 and K3; injury model panel samples UVA were set in 3 parallel groups to reduce error, labeled UVA1, UVA2, UVA3; 3 parallel groups of freeze-dried powder (sample group) (1 mg/mL) M are arranged to reduce errors, and marked as M1, M2 and M3; succinic acid (sample group) (1 mg/mL) H was set in 3 parallel groups to reduce errors, labeled H1, H2, H3. In order to ensure the accuracy and reliability of the data quality, the experiment firstly carries out quality control analysis and correlation analysis on the sequencing data, and the result shows that the sequencing data has good quality and high repeatability. Next, differential analysis was performed on gene expression using DESeq, and statistics of differential genes between groups are shown in table 2.
TABLE 2 statistical results of differential genes
In table 2, K is the blank control group, UVA is the damage model group, M is the lyophilized powder (sample group), and H is succinic acid (sample group). In Table 2, the comparison of the K group and the UVA group is shown by taking K vs UVA as an example, the comparison is obtained by carrying out statistical analysis on the results of K1, K2, K3, UVA1, UVA2 and UVA3, the total difference is 2495, 1729 is up-regulated, 766 is down-regulated, and the damage model is constructed.
UVA vs M is a comparison of the injury model group and the lyophilized powder (sample group), the total number of differences was 262, up-regulated 57, down-regulated 205, indicating that the lyophilized powder sample has an effect on UVA injury.
UVA vs H is the result of a comparison of the injury model group and succinic acid (sample group), the total number of differences was 1898, up-regulated 499 and down-regulated 1399.
M vs H is the result of comparing the freeze-dried powder (sample group) with succinic acid (sample group), the total difference is 243, the up-regulation is 148, the down-regulation is 95, and the influence of the freeze-dried powder sample on cells is different from that of succinic acid on cells.
K vs M is the result of comparison between the blank control group and the freeze-dried powder (sample group), the total difference is 2308, 1313 is up-regulated, 995 is down-regulated, and the freeze-dried powder sample has an effect on normal cells which are not damaged by UVA.
K vs H is the result of comparison of the blank and succinic acid (sample) groups, the total number of differences is 3739, upregulated 1749, downregulated 1997, indicating that succinic acid has an effect on normal cells not damaged by UVA.
Next, GO function enrichment analysis is performed on the differential genes, the result is shown in fig. 1, fig. 1 is a GO function enrichment analysis chart of different differential genes, and note that: the method comprises the steps of sequentially comparing a blank control group with a damage model group, a damage model group with a freeze-dried powder group, and a damage model group with a succinic acid group from top to bottom, wherein the abscissa is-log 10 (P value), and the ordinate is GO Term.
In fig. 1, P value is a statistical test variable representing the significance of the difference, and the abscissa is-log 10 (P value), with larger values the more significant the difference. CC in ordinate: a cellular component; BP: a biological process; MF: molecular functions, distinguished by different colors.
The results of the differential gene KEGG pathway enrichment analysis are shown in FIG. 2, which shows that compared with the damage model group, the lyophilized powder and the succinic acid group have the differential gene mainly enriched in the TNF signal pathway, and the TNF SIGNALING PATHWAY pathway in FIG. 2 can regulate and control the expression of the chemotactic factor after being activated, so that the expression of the chemotactic factor can be inhibited when the pathway is inhibited. Among them, the chemokine CXCL2 changes significantly. In fig. 2, a blank group, a model group, a freeze-dried powder group, a damage model group, and a succinic acid group are sequentially arranged from top to bottom, wherein the abscissa is-log 10 (P value), and the ordinate is KEGG PATHWAY.
1Mg/mL, 0.5mg/mL, 0.25mg/mL of freeze-dried powder and 1mg/mL, 0.5mg/mL and 0.25mg/mL of succinic acid can reduce oxidative damage of UVA to human skin fibroblasts, wherein the 1mg/mL of freeze-dried powder and 1mg/mL of succinic acid have the best effect. After 1mg/mL of lyophilized powder and 1mg/mL of succinic acid are detected by high-throughput transcriptome sequencing, compared with a model group, the differential gene is mainly enriched in a TNF signal channel, wherein the expression quantity of the chemokine CXCL2 is obviously reduced, and the expression of the chemokine CXCL2 is presumed to be inhibited, so that the human skin fibroblast is further anti-aging.
FIG. 3 shows the results of the effect of different chemokines in different groups, FIG. 3 shows that Control is a blank, UVA is a damage model, UVA+ MFY is a lyophilized powder group, UVA+SA is a succinic acid group, and FIG. 3 shows that CXCL1 and CXCL6 are affected differently in different groups, and it can be seen that the effect of these two chemokines is less.
As can be seen from fig. 2 and 3, the differential genes are mainly enriched in TNF signaling pathways compared to the injury model group, where the reduction in chemokine CXCL2 expression is more pronounced, with the chemokines CXCL1, CXCL6 being less affected in the different groups.
(3) Elastin assay and TGF-beta 1 content assay
(3.1) Fibroblasts were seeded at a 2.2X10 5 count/well seeding density on 6 well plates and incubated overnight in an incubator (37 ℃,5% CO 2). UVA irradiation: depending on the test group, the group requiring UVA irradiation is subjected to 30J/cm 2 of UVA irradiation. And detecting by a Western blot method.
(3.2) Fibroblasts were seeded at a seeding density of 2.2X10 5 pieces/well in 6-well plates and cultured in an incubator (37 ℃ C., 5% CO 2). RNA is extracted, reverse transcribed into cDNA and detected by fluorescent quantitative PCR. Mapping was performed using GRAPHPAD PRISM and the results are expressed in mean±sd.
Table 3 set of groups
Experimental results
1. 2% Sturgeon collagen-type I collagen test results
TABLE 4 collagen type I test results
Compared with the blank control group, the content of the type I collagen of the negative control group is obviously reduced, which proves that the test stimulation condition is effective. The positive control group had a significantly increased type I collagen content compared to the negative control group. Compared with the negative control group, the improvement rate of 2% sturgeon collagen and rice fermentation broth (sample group 1) is 76.73%.
2. 2% Sturgeon collagen-elastin test results
TABLE 5 elastin assay results
Compared with a blank control group, the elastin content of the negative control group is obviously reduced, which proves that the test stimulation condition is effective. The positive control group had significantly higher elastin content than the negative control group. Compared with the negative control group, the improvement rate of the collagen of the sturgeon and the rice fermentation liquor is improved by 68.99 percent.
3. 2% Sturgeon collagen TGF-beta 1 detection result
TABLE 6 TGF-. Beta.1 assay results
Compared with a blank control group, the TGF-beta gene expression of the negative control group is significantly down-regulated, which proves that the test stimulation condition is effective. The positive group had TGF-. Beta.1 compared to the negative control group. The gene expression level was significantly up-regulated. Compared with the negative control group, the improvement rate of TGF-beta 1 of 2% sturgeon collagen and rice fermentation liquor (sample group 1) is improved by 43.66%
Application example provides the application of the photodamage-resistant rice fermentation broth mixture freeze-dried powder of the examples 1-3 in cosmetics, and specifically provides an emulsion containing photodamage-resistant rice fermentation broth mixture freeze-dried powder;
TABLE 7 proportions of raw material components of examples 1-3 and comparative examples
The method for preparing the emulsion containing the photodamage-resistant rice fermentation liquor mixture freeze-dried powder comprises the following steps:
(1) Mixing the components of component A in Table 3, stirring at 40deg.C for 25min at 200rpm to form an oil phase;
(2) Mixing the components of component B in Table 3, stirring at 40deg.C for 15min at 400rpm to form a water phase;
(3) Mixing and stirring the oil phase and the water phase at 40 ℃ for 15min, adding essence, bromopol and propylparaben, stirring again at 40 ℃ for 20min at 300rpm, and passing through a nanometer homogenizer for 5 times to obtain an essence emulsion frame.
(4) When in use, the D phase is uniformly mixed with the essence emulsion frame.
Clinical experiments on fish tail lines of emulsions containing photodamage-resistant rice broth mixture lyophilized powder
Subjects were 40 chinese healthy sensitive myofemales, divided into 4 groups of 10, each group tested the emulsions of the application, comparative application, between 27 and 50 years of age, with an average age of 35 years, and subjects informed with written consent. The subject would like to coordinate and understand the necessity and duration of control in order to follow the protocol established by the clinical trial center. The testers all have obvious fish tail lines, the eye trial application example of the testers or the emulsion of the comparison application example is used once in the morning and evening for 28 days, the PRIMOS clinical research system is adopted to measure the trial effect, the average value of 10 subjects in each group is calculated after the test data are recorded, and the obtained results are shown in Table 8.
The test results are shown in Table 8.
TABLE 8 clinical study results
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In table 8, all improvement rates refer to the "decrease rate" of D0 at the beginning. The average depth of the corner wrinkles, the number of the corner wrinkles, the area of the corner wrinkles, the length of the corner wrinkles, and the volume of the corner wrinkles are all indexes obtained by facial image acquisition and wrinkle analysis by Primos. As can be seen in Table 8, the average values from day 0 (D0) to day 14 (D14) and day 28 (D28). After 28 days, the emulsion of the freeze-dried powder of the rice fermentation liquid mixture with photodamage resistance can effectively reduce the wrinkle depth and remarkably remove the fish tail.
The previous description of the embodiments is provided to facilitate a person of ordinary skill in the art in order to make and use the present invention. It will be apparent to those skilled in the art that various modifications can be readily made to these embodiments and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above-described embodiments, and those skilled in the art, based on the present disclosure, should make improvements and modifications without departing from the scope of the present invention.
Claims (10)
1. A preparation method of photodamage-resistant rice fermentation liquor mixture freeze-dried powder is characterized by comprising the following steps:
(1) Dissolving a mixture consisting of rice fermentation liquor and sturgeon collagen and mannitol in water for injection, adding active carbon, uniformly stirring, and filtering out a primary filtrate by using filter cloth;
(2) Filtering the primary filtrate obtained in the step (1) by using a microporous sterilization filter to obtain secondary filtrate, canning the secondary filtrate into a sterilized container, and transferring the container into a freeze dryer under the aseptic condition;
(3) The container filled with the secondary filtrate is primarily frozen at the temperature of minus 20 ℃ to minus 10 ℃, and then is further cooled to the temperature of minus 50 ℃ to minus 40 ℃ and frozen for 2 to 3 hours, so that the container is completely frozen; then the freezing temperature is stably increased to-25 to-15 ℃, vacuumizing is carried out, the temperature is increased to-5 to 0 ℃ within 10 to 12 hours, and the temperature is maintained for 4 to 8 hours at the temperature of-5 to 0 ℃; and heating to 35-38 ℃ within 2-3 hours, maintaining the temperature and extreme vacuum to carry out secondary analysis and dehydration for 3-4 hours, and exhausting after dehydration is finished to obtain the photodamage-resistant rice fermentation liquor mixture freeze-dried powder.
2. The method for preparing photodamage resistant rice fermentation broth mixture freeze-dried powder according to claim 1, wherein in the step (1), the mixture consisting of rice fermentation broth and sturgeon collagen is mixed with mannitol according to the volume ratio of the mixture to mannitol of 1-3:1, preferably 3:2, the volume ratio of the sum of the volumes of the mixture and mannitol to the volume ratio of the water for injection is 1:2-4.
3. The method for preparing photodamage resistant rice fermentation broth mixture freeze-dried powder according to claim 1, wherein in the step (1), the mass ratio of the rice fermentation broth to the sturgeon collagen in the mixture consisting of the rice fermentation broth and the sturgeon collagen is 1-4:1, preferably 2:1.
4. The method for preparing photodamage resistant rice fermentation broth mixture freeze-dried powder according to claim 1, wherein in the step (1), the mesh number of the filter cloth is 800-5000 mesh; in the step (2), the pore diameter of the microporous sterilization filter is 0.22-0.3 μm.
5. Photodamage-resistant rice fermentation broth mixture freeze-dried powder obtained based on the preparation method of any one of claims 1-4.
6. The use of photodamage resistant rice broth mixture lyophilized powder of claim 5 in cosmetics.
7. The use according to claim 6, wherein the photodamage resistant rice fermentation broth mixture lyophilized powder is formed into a related cosmetic product with a cosmetically acceptable carrier or excipient.
8. The use according to claim 7, wherein the cosmetic product is a facial cleanser, a lotion, a skin cleansing lotion, a balance water, an emulsion, a cream, a mask or a bath.
9. An essence emulsion containing photodamage-resistant rice fermentation liquor mixture freeze-dried powder is characterized by comprising the following components
The rice fermentation liquor mixture freeze-dried powder is photodamage-resistant rice fermentation liquor mixture freeze-dried powder prepared by the preparation method of claim 1.
10. The process for preparing the essential emulsion of claim 9, comprising the steps of:
(1) Mixing the components of the component A, stirring for 15-35 min at 20-50 ℃ and stirring speed of 150-300rpm to form an oil phase;
(2) Mixing the components of the component B, stirring for 10-25 min at 20-50 ℃ and the stirring speed is 300-500 rpm to form a water phase;
(3) Mixing and stirring the oil phase and the water phase at 20-50 ℃ for 5-20 min, adding essence, bromopol and propylparaben, stirring again at 20-50 ℃ for 10-30 min at 200-400 rpm, and passing through a nano homogenizer for 3-8 times to obtain the essence emulsion frame.
(4) When in use, the phase D and the essence emulsion framework are uniformly mixed, and the essence emulsion containing the photodamage-resistant rice fermentation liquor mixture freeze-dried powder is obtained.
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| CN202311409701.6A CN117959226A (en) | 2023-10-27 | 2023-10-27 | Photodamage-resistant rice fermentation liquor mixture freeze-dried powder and preparation method and application thereof |
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