Use of rice ferment filtrate freeze-dried powder in the preparation of products for repairing skin damage

Abstract

The present application relates to the application of rice fermentation broth freeze-dried powder in the preparation of products for repairing skin damage. Compared with the prior art, the present application first discovers that the rice fermentation broth freeze-dried powder has a certain repairing effect on human skin fibroblast HES damage or HaCaT cell damage, the effective concentration is low, and there is no toxic side effect. Through the UV-induced skin cell damage model, it is found that the rice fermentation broth freeze-dried powder can effectively resist oxidative damage and reduce the excessive generation of ROS caused by UV. The present application studies the mechanism of the repairing effect of rice fermentation broth freeze-dried powder on human skin fibroblast HES damage or HaCaT cell damage, and expands the new application field of rice fermentation broth freeze-dried powder.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of daily chemicals, and particularly relates to application of rice fermentation liquor freeze-dried powder in preparation of a product for repairing skin damage. BACKGROUND

[0002] According to the wavelength of ultraviolet rays, UV is divided into UVA (320-400 nm), UVB (280-320 nm) and UVC (100-280 nm). The irradiation of ultraviolet rays is easy to cause damage to the skin. When the ultraviolet rays act on the skin for a long time, the activity of matrix metalloproteinases (MMPs) is excessively excited, resulting in a large amount of collagen and elastin in the dermis layer of the skin being degraded, so that the collagen fibers lose elasticity and become relaxed and weak, and finally form wrinkles. At the same time, when the skin is excessively exposed to ultraviolet rays, the structure of the extracellular matrix is destroyed, and the skin immune function is also reduced, the skin tissue cell activity is weakened, and the skin aging is caused. At the same time, the ultraviolet rays can also enhance the activity of the pigment cells in the epidermis layer, thereby causing abnormal color of the facial skin. Among them, UVA in the ultraviolet rays has strong penetrating power, can penetrate the epidermis layer of the skin and reach the dermis layer, thereby destroying the collagen and elastin in the dermis layer, affecting the tightness and elasticity of the skin, and causing photoaging skin aging such as pigmentation, wrinkles and relaxation. And UVB in the ultraviolet rays can penetrate the epidermis layer of the skin and directly damage the skin cells, produce a large amount of inflammation, cause skin tanning, sunburn, peeling and pigmentation; at the same time, UVB can destroy the moisturizing ability of the skin, so that the skin becomes rough and wrinkled. Therefore, repairing ultraviolet skin damage is an important means to slow down aging.

[0003] The existing components for repairing skin damage are mainly some substances playing an antioxidant role, such as superoxide dismutase (SOD), glutathione peroxidase (GSH-PX) and the like, which can remove superoxide anion free radicals and delay the aging process of the body.

[0004] At present, the application of rice fermentation liquor in the skin care field generally focuses on its effect on the stability of the microbial flora on the skin surface, and there is no research on which substance in the rice fermentation liquor plays a role, and there is no excessive focus on whether the rice fermentation liquor can directly repair the skin cell damage caused by ultraviolet rays. SUMMARY

[0005] The purpose of the present application is to provide application of rice fermentation liquor freeze-dried powder in preparation of a product for repairing skin damage.

[0006] The purpose of the present application can be achieved by the following technical solutions.

[0007] The present application provides a use of rice fermentation broth freeze-dried powder in the preparation of a product for repairing skin damage, and the rice fermentation broth freeze-dried powder is added into the product for repairing skin damage, and the concentration of the rice fermentation broth freeze-dried powder in the product is 1.25-10 mg / mL.

[0008] In one embodiment of the present application, the rice fermentation broth freeze-dried powder is prepared from rice fermentation broth, and the rice fermentation broth freeze-dried powder contains succinic acid and choline.

[0009] In one embodiment of the present application, preferably, the concentration of the rice fermentation broth freeze-dried powder in the product is 5-10 mg / mL.

[0010] In one embodiment of the present application, the skin damage is cell damage caused by ultraviolet rays.

[0011] Further, the skin damage refers to skin damage caused by UVA or UVB irradiation.

[0012] In one embodiment of the present application, the cell damage caused by ultraviolet rays is damage to human keratinocyte HaCat and / or human skin fibroblast HES caused by ultraviolet rays.

[0013] In one embodiment of the present application, the product is a product having a promoting effect on the proliferation of melanoma cells B16.

[0014] In one embodiment of the present application, the product is a product capable of reducing the generation of ROS caused by UV and repairing damage to human skin fibroblast HES or HaCaT cells. In one embodiment of the present application, the product is selected from a pharmaceutical product or a cosmetic product.

[0015] In one embodiment of the present application, the product further comprises a carrier, an excipient or a diluent allowed in a pharmaceutical product or a cosmetic product.

[0016] In one embodiment of the present application, the product is an emulsion, a cream, a capsule, a tablet, an oral preparation, a microcapsule preparation or an injection.

[0017] In the present application, the rice fermentation broth is obtained by fermenting rice slurry with yeast. In one preferred embodiment of the present application, the rice fermentation broth is prepared by fermentation and enrichment processes using rice from Akita, Japan, and the yeast used is saccharomyces veronae, and the rice fermentation broth is rich in choline and succinic acid.

[0018] The present application provides a feasible method for preparing rice fermentation broth, which is as follows:

[0019] (1) rice powder is heated, stirred, and becomes rice slurry, and beta-glucanase is added into the rice slurry, the enzyme is added in an amount of 0.001%-0.5% of the mass of the rice slurry, the enzyme activity is 150-200 U / g, and enzyme hydrolysis is carried out, so that rice slurry enzyme hydrolysate is obtained;

[0020] (2) the yeast is inoculated into the rice slurry enzyme hydrolysate, and fermentation is carried out under the following conditions: the fermentation temperature is 20-40 DEG C, the fermentation time is 24-48 hours, and the pH during the fermentation process is controlled to be between 6 and 6.5, so that rice fermentation liquor is obtained after fermentation.

[0021] (3) the rice fermentation liquor after fermentation is concentrated through reverse osmosis technology, active substances are efficiently enriched, sterilization is carried out after heating, cooling and filtration are carried out, so that concentrated rice fermentation liquor, i.e., rice fermentation liquor containing active substances, is obtained;

[0022] In the present application, the rice fermentation liquor freeze-dried powder is prepared from rice fermentation liquor, and can be obtained by using a conventional preparation method. In the present application, the rice fermentation liquor is used in the form of freeze-dried powder, and the freeze-dried powder can maximize the enrichment of active ingredients (especially succinic acid and choline) and has higher stability.

[0023] The present application provides a feasible preparation method of rice fermentation liquor freeze-dried powder, which is as follows: the obtained rice fermentation liquor is placed in a freeze dryer, vacuum freeze-drying is carried out, the freezing temperature is-65 DEG C to-80 DEG C, the vacuum degree is 30 Pa to 50 Pa, and the freezing time is 48 h, so that the rice fermentation liquor freeze-dried powder is obtained. Since the rice fermentation liquor contains succinic acid and choline, the rice fermentation liquor freeze-dried powder in the present application is a concentrated freeze-dried powder of rice fermentation liquor, and therefore, the rice fermentation liquor freeze-dried powder also contains succinic acid and choline.

[0024] The present application finds through experimental research that the rice fermentation liquor freeze-dried powder with a concentration of 1.25-10 mg / ml is a safe concentration for human keratinocyte HaCat. The rice fermentation liquor freeze-dried powder with a concentration of 1.25-10 mg / ml is a safe concentration for human skin fibroblast HES, and it is shown that the rice fermentation liquor freeze-dried powder has a significant effect of promoting the proliferation of human skin fibroblast HES. The rice fermentation liquor freeze-dried powder with a concentration of 1.25-10 mg / ml is a safe concentration for mouse melanoma cell B16, and it is shown that the rice fermentation liquor freeze-dried powder has a significant effect of promoting the proliferation of mouse melanoma cell B16. Further research shows that the rice fermentation liquor freeze-dried powder has a certain repairing effect on human skin fibroblast HES damage or HaCaT cell damage.

[0025] Compared with the prior art, the present application has the following beneficial effects:

[0026] (1) The application first discovers that rice fermentation liquor freeze-dried powder has certain repairing effect on human skin fibroblast HES injury or HaCaT cell injury, has low effective concentration, and has no toxic side effects.

[0027] (2) The application discovers that the rice fermentation liquor freeze-dried powder can effectively resist oxidative damage and reduce excessive ROS production caused by UV through a UV-induced skin cell injury model.

[0028] (3) The application studies the mechanism of the rice fermentation liquor freeze-dried powder on the repairing effect of human skin fibroblast HES injury or HaCaT cell injury, and expands the new application field of the rice fermentation liquor freeze-dried powder. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 ROS fluorescence intensity diagram of sample preventing UVA injury HES cells,

[0030] Among them, A: blank control group; B: injury model group; C: vitamin C positive control; D: 10 mg / mL rice freeze-dried powder; E: 5 mg / mL rice freeze-dried powder; F: 2.5 mg / mL rice freeze-dried powder; G: 1 mg / mL succinic acid; H: 0.5 mg / mL succinic acid; I: 0.25 mg / mL succinic acid;

[0031] Figure 2 ROS fluorescence intensity columnar diagram of sample preventing UVA injury HES cells. DETAILED DESCRIPTION

[0032] The application will be described in detail below in combination with the drawings and specific embodiments.

[0033] Embodiment 1

[0034] The embodiment provides a preparation method of rice fermentation liquor freeze-dried powder, which comprises the following steps:

[0035] (1) rice powder is heated, stirred and added with water to become 30 wt % rice slurry, β-glucanase is added into the rice slurry, the enzyme addition amount is 0.001%-0.5% of the mass of the rice slurry, the enzyme activity is 150-200 U / g, and enzyme hydrolysis is carried out to obtain rice slurry enzyme hydrolysate;

[0036] (2) the yeast strain is inoculated into the rice slurry enzyme hydrolysate, and the fermentation conditions are as follows: the fermentation temperature is 20-40 ℃, the fermentation time is 24-48 hours, the pH in the fermentation process is controlled to be between 6-6.5, and the rice fermentation liquor is obtained after fermentation;

[0037] (3) the rice fermentation liquor after fermentation is concentrated through reverse osmosis technology, active substances are efficiently enriched, sterilization is carried out through heating, cooling and filtration to obtain the concentrated rice fermentation liquor, i.e. the rice fermentation liquor containing active substances;

[0038] (4) The obtained rice fermentation broth is placed in a freeze dryer, vacuum freeze-dried, the freezing temperature is -65℃ to -80℃, the vacuum degree is 30 to 50 Pa, and the freezing time is 48 h, so that the rice fermentation broth freeze-dried powder is obtained.

[0039] The content of succinic acid and choline in the rice fermentation broth prepared in step (2) of the embodiment is detected by high performance liquid chromatography (HPLC).

[0040] The HPLC conditions of succinic acid are as follows: the chromatographic column is Kromasil C18 chromatographic column (150 mm × 4.6 mm, 5 μm), the mobile phase is methanol and 0.02 mol / L KH2PO4 buffer (volume ratio is 7:93), the column temperature is 30℃, the flow rate is 0.5 mL / min, and the detection wavelength is 214 nm.

[0041] The HPLC conditions of choline are as follows: the chromatographic column is Kromasil 100-5-SIL silica gel column, the column temperature is 30℃, the mobile phase is n-hexane: isopropyl alcohol: water (volume ratio is 4:80:16), the flow rate is 0.5 mL / min, and the detection wavelength is 207 nm.

[0042] The content of succinic acid and choline in the rice fermentation broth obtained in the embodiment is shown in Table 1.

[0043] Table 1 Content of succinic acid and choline in rice fermentation broth obtained in the embodiment

[0044]

[0045] In Table 1, the preparation processes of rice fermentation broth samples No. 1, 2, 3, 4 and 5 are shown in Table 2 respectively.

[0046] Table 2 Preparation processes of rice fermentation broth samples No. 1, 2, 3, 4 and 5

[0047]

[0048] It can be seen that the addition amount of enzyme, enzyme activity, fermentation temperature and fermentation time have an influence on the content of succinic acid and choline in the finally obtained rice fermentation broth sample, but as long as the preparation method in Example 1 is used, the rice fermentation broth containing succinic acid and choline can be obtained, and preferably the rice fermentation broth containing succinic acid and choline obtained by the preparation method of sample No. 3 in Example 1 is used.

[0049] The rice fermentation broth freeze-dried powder is obtained by placing the obtained rice fermentation broth into a freeze dryer and vacuum freeze-drying, and the operation can be performed under the conditions of a freezing temperature of-65℃ to-80℃, a vacuum degree of 30 to 50 Pa, and a freezing time of 48 h. In this embodiment, the rice fermentation broth freeze-dried powder is obtained by vacuum freeze-drying the sample with sample number 3 prepared in step (3) under the conditions of a freezing temperature of-70℃, a vacuum degree of 40 Pa, and a freezing time of 48 h. The active ingredients in the rice fermentation broth are not destroyed by freeze-drying. Since the rice fermentation broth contains succinic acid and choline, the rice fermentation broth freeze-dried powder in this application is a concentrated freeze-dried powder of the rice fermentation broth, and therefore, the rice fermentation broth freeze-dried powder also contains succinic acid and choline.

[0050] Effect Example 1

[0051] In this effect example, the effect of rice fermentation broth freeze-dried powder with different concentrations on the survival rate of human keratinocytes HaCat is determined by the CCK-8 method.

[0052] The rice fermentation broth freeze-dried powder prepared from the rice fermentation broth sample with sample number 3 in Example 1 (abbreviated as rice freeze-dried powder in the drawing) is used for experimental research.

[0053] The survival rate of human keratinocytes HaCaT with different concentrations of rice fermentation broth freeze-dried powder is tested, and the specific test steps are as follows:

[0054] (1) Cell inoculation: HaCaT cells are inoculated into 24-well culture plates at a cell density of 2-3×10 5 per well, and incubated in a CO2 cell incubator for 24 h.

[0055] (2) Sample addition: The liquid in the hole plate is sucked out with a pipette, and the rice fermentation broth freeze-dried powder sample solution prepared with serum-free cell culture medium (0 mg / mL, 1.25 mg / mL, 2.5 mg / mL, 5 mg / mL, 10 mg / mL) is added to the hole plate, and incubated for 24 h as the experimental group; the blank control group is not added with rice fermentation broth freeze-dried powder, but only with serum-free medium.

[0056] (3) Cell survival rate detection: after incubation, the above cell culture medium after incubation is sucked out with a pipette, CCK8 detection solution (CCK8: high glucose medium = 1:10) is added to the hole plate, and incubated for 2-4 h. After the CCK8 detection solution is completely reacted, the hole plate is taken out from the cell incubator and the absorbance of the liquid in the hole plate at 450 nm is detected by an enzyme marker. Among them, the absorbance of the blank control group is the absorbance of the CCK8 detection solution after simple incubation.

[0057] The cell survival rate calculation formula is as follows:

[0058] HaCat cell survival rate (%) = absorbance of experimental group / absorbance of blank control group x 100%

[0059] The results of the effect of rice fermentation broth freeze-dried powder of different concentrations on the survival rate of human keratinocytes HaCat are shown in Table 3.

[0060] Table 3 Effect of rice fermentation broth freeze-dried powder of different concentrations on the survival rate of human keratinocytes HaCat

[0061]

[0062] *: p <0.05; **: p <0.01

[0063] The results in Table 3 show that when the concentration of rice fermentation broth freeze-dried powder is 10 mg / mL, the survival rate of human keratinocytes HaCat is 93.35%, and the result is statistically significant. When the concentration is 1.25-10 mg / ml, the survival rate of human keratinocytes HaCat is 90% or more, indicating that rice fermentation broth freeze-dried powder with a concentration of 1.25-10 mg / ml is a safe concentration for human keratinocytes HaCat.

[0064] Effect Example 2

[0065] In this effect example, the effect of rice fermentation broth freeze-dried powder of different concentrations on the survival rate of human skin fibroblasts HES was determined by CCK-8 method.

[0066] The rice fermentation broth freeze-dried powder prepared from sample No. 3 of Example 1 was used for experimental study.

[0067] The effect of rice fermentation broth freeze-dried powder of different concentrations on the survival rate of human skin fibroblasts HES was tested, and the specific test steps were as follows:

[0068] (1) Cell inoculation: HES cells were inoculated in 24-well culture plates at a cell density of 2-3 x 10 5 per well, and incubated in a CO2 cell incubator for 24 h.

[0069] (2) Sample addition: The liquid in the well plate was removed with a pipette, and rice fermentation broth freeze-dried powder sample solution (0 mg / mL, 1.25 mg / mL, 2.5 mg / mL, 5 mg / mL, 10 mg / mL) prepared with serum-free cell culture medium was added to the well plate, and incubated for 24 h as the experimental group; the blank control group was not added with rice fermentation broth freeze-dried powder, but only with serum-free medium.

[0070] (3) Cell survival rate detection: after the incubation is completed, the culture medium after the incubation of the above cells is sucked out with a pipette, CCK8 detection liquid (CCK8: high-sugar culture medium = 1:10) is added to the hole plate, and incubation is continued for 2-4 h. After the CCK8 detection liquid reaction is complete, the hole plate can be taken out of the cell incubator and the absorbance of the liquid in the hole plate at 450 nm is detected by an enzyme label instrument. Among them, the absorbance of the blank control group is the absorbance of the CCK8 detection liquid itself after incubation.

[0071] Among them, the cell survival rate calculation formula is as follows:

[0072] HES cell survival rate (%) = absorbance of experimental group / absorbance of blank control group x 100%

[0073] The results of the effect of rice fermentation liquid freeze-dried powder of different concentrations on the survival rate of human skin fibroblast cells HES are shown in Table 4.

[0074] Table 4 Effect of rice fermentation liquid freeze-dried powder of different concentrations on the survival rate of human skin fibroblast cells HES

[0075]

[0076] p Less than 0.05; **: p Less than 0.01

[0077] The results in Table 4 show that the survival rate of human skin fibroblast cells HES is 100% and above in the case of a concentration of 1.25-10 mg / ml, indicating that the rice fermentation liquid freeze-dried powder with a concentration of 1.25-10 mg / ml is a safe concentration for human skin fibroblast cells HES, and that the rice fermentation liquid freeze-dried powder has a significant effect on promoting the proliferation of human skin fibroblast cells HES.

[0078] Effect Example 3

[0079] This effect example studies the effect of rice fermentation liquid freeze-dried powder of different concentrations on the survival rate of mouse melanoma cells B16

[0080] The rice fermentation liquid freeze-dried powder prepared from sample No. 3 of Example 1 is used for test research.

[0081] The effect of rice fermentation liquid freeze-dried powder of different concentrations on the survival rate of mouse melanoma cells B16 is tested, and the specific test steps are as follows:

[0082] (1) Cell inoculation: mouse melanoma cells B16 are inoculated in the holes of a 24-hole culture plate at a cell density of 2-3 x 10 5 / hole, and are placed in a CO2 cell incubator for incubation for 24 h.

[0083] ​(2) Sample addition: The liquid in the well plate was aspirated with a pipette, and the sample solution of rice fermentation broth freeze-dried powder prepared with serum-free cell culture medium (0 mg / mL, 1.25 mg / mL, 2.5 mg / mL, 5 mg / mL, 10 mg / mL) was added to the well plate, and the culture was continued for 24 h, which was the experimental group; the blank control group was not added with rice fermentation broth freeze-dried powder, but only with serum-free medium.

[0084] (3) Cell survival rate detection: after incubation, the culture medium after cell incubation was aspirated with a pipette, CCK8 detection solution (CCK8: high-sugar medium = 1:10) was added to the well plate, and incubation was continued for 2-4 h. After the CCK8 detection solution was completely reacted, the well plate was taken out from the cell incubator and the absorbance of the liquid in the well plate at 450 nm was detected by an enzyme marker. Among them, the absorbance of the blank control group was the absorbance of the CCK8 detection solution after incubation alone.

[0085] The cell survival rate calculation formula is as follows:

[0086] The survival rate of mouse melanoma cells B16 (%) = experimental group absorbance / blank control group absorbance x 100%

[0087] The results of the effect of different concentrations of rice fermentation broth freeze-dried powder on the survival rate of mouse melanoma cells B16 are shown in Table 5.

[0088] Table 5 Effect of different concentrations of rice fermentation broth freeze-dried powder on the survival rate of mouse melanoma cells B16

[0089]

[0090] *: p Less than 0.05; **: p Less than 0.01

[0091] As can be seen from Table 5, the survival rate of mouse melanoma cells B16 is 100% or more at a concentration of 1.25-10 mg / ml, indicating that the concentration of 1.25-10 mg / ml of rice fermentation broth freeze-dried powder is a safe concentration for mouse melanoma cells B16, and it is shown that the rice fermentation broth freeze-dried powder has a significant effect on promoting the proliferation of mouse melanoma cells B16.

[0092] Effect Example 4

[0093] This effect example studies the effects of rice fermentation broth freeze-dried powder and succinic acid on long-wave ultraviolet (UVA) damaged human skin fibroblasts HES

[0094] The rice fermentation broth freeze-dried powder prepared from sample No. 3 of Example 1 and succinic acid were respectively tested and studied.

[0095] The effect of rice fermentation broth freeze-dried powder on the survival rate of human skin fibroblasts (HES) damaged by long-wave ultraviolet (UVA) radiation was determined by the CCK-8 assay.

[0096] The test method is as follows:

[0097] Human skin fibroblasts (HES) without rice fermentation broth lyophilized powder were placed in a culture medium and irradiated with UVA (UVA radiation dose of 2 J / cm²). 2 After that, it becomes a model group;

[0098] Human skin fibroblasts (HES) without the addition of rice fermentation broth lyophilized powder were placed in a culture medium. Cells not exposed to UVA served as the control group.

[0099] Human skin fibroblasts (HES) were cultured with different concentrations of rice fermentation broth lyophilized powder (2.5 mg / mL, 5 mg / mL, 10 mg / mL) for 24 hours, and then irradiated with UVA (UVA radiation dose of 2 J / cm²). 2 After that, different concentrations of rice fermentation broth lyophilized powder (2.5 mg / mL, 5 mg / mL, 10 mg / mL) were added and cultured for 24 hours to form the rice fermentation broth lyophilized powder experimental group.

[0100] Human skin fibroblasts (HES) were cultured with different concentrations of succinic acid (1 mg / mL, 0.5 mg / mL, and 0.25 mg / mL) for 24 hours before being irradiated with UVA (UVA radiation dose of 2 J / cm²). 2 After that, different concentrations of succinic acid (1 mg / mL, 0.5 mg / mL, 0.25 mg / mL) were added and cultured for 24 hours to form the succinic acid experimental group;

[0101] Human skin fibroblasts (HES) pre-treated with different concentrations of choline (3.125 mg / ml, 6.25 mg / ml, and 12.5 mg / ml) for 24 hours were then irradiated with UVA (UVA radiation dose of 2 J / cm²). 2 After that, different concentrations of choline (3.125 mg / ml, 6.25 mg / ml, 12.5 mg / ml) were added and cultured for 24 hours to form the choline experimental group;

[0102] The culture medium used in the model group, blank group, and rice fermentation broth freeze-dried powder experimental group was conventional DMEM high-glucose medium, which consists of amino acids, vitamins / cofactors, inorganic salts, phenol red, etc.

[0103] The survival rate of human skin fibroblasts (HES) in different experimental groups was determined using the CCK-8 assay.

[0104] The formula for calculating the survival rate of human skin fibroblasts HES is:

[0105] HES cell survival rate (%) = (absorbance of the experimental group - absorbance of the blank group) / (absorbance of the model group - absorbance of the blank group) x 100%

[0106] The effect of rice fermentation broth freeze-dried powder on the survival rate of UVA-damaged human skin fibroblasts is shown in Table 6.

[0107] Table 6 Effect of rice fermentation broth freeze-dried powder on the survival rate of UVA-damaged fibroblasts

[0108]

[0109] Compared with the blank group,*** p P<0.001; compared with the model group,# p P<0.05; and p P<0.001

[0110] The experimental results show that the cell survival rate after UVA irradiation decreased to 76.70%, which was statistically significant compared with the blank group, indicating that the UVA-induced cell photodamage model was successfully constructed.

[0111] After pre-adding rice fermentation broth freeze-dried powder for 24 h, the cells were irradiated, and then rice fermentation broth freeze-dried powder was added for 24 h. The cell survival rate was increased to 99.34% and 93.75% at concentrations of 10 mg / mL and 5 mg / mL, respectively, which was statistically significant compared with the model group, and had no statistical difference compared with the blank group. Therefore, 10 mg / mL and 5 mg / mL rice fermentation broth freeze-dried powder had a certain repair effect on UVA-damaged human skin fibroblasts HES.

[0112] Effect Example 5

[0113] This effect example studies human skin fibroblasts HES in the blank control group, the damage model group, the positive control group, and the different sample groups through a cell ROS inhibition experiment to verify the repair effect of rice fermentation broth freeze-dried powder on UVA-damaged human skin fibroblasts HES.

[0114] The specific operation steps of the cell ROS inhibition experiment are as follows:

[0115] (1) Cell inoculation: human skin fibroblasts HES cells were inoculated in 24-well culture plates at a cell density of 2~3 x 10 5 cells / well, and then placed in a CO2 cell incubator for 24 h.

[0116] (2) According to the experimental grouping, the test substance is added into the cells for 24 h, and the experimental groups are as follows: serum-free medium (blank control group, which is not subjected to UVA irradiation later), serum-free medium (damage model group, which is subjected to UVA irradiation later), vitamin C (200 ug / mL, positive control group), serum-free medium respectively prepared with rice freeze-dried powder (10, 5, 2.5 mg / mL, sample group), succinic acid (1, 0.5, 0.25 mg / mL, sample group).

[0117] (3) Except for the blank control group, the rest of the cells are subjected to UVA irradiation (2 J / cm 2 ).

[0118] (4) The culture solution is discarded, and the cells are washed and fixed, and the working concentration of the active oxygen fluorescent probe DCFH-DA reagent is added into the cells for co-incubation according to the reagent operation instruction.

[0119] (5) After the incubation is completed, the cells are photographed under an inverted fluorescence microscope, and the fluorescence intensity is counted.

[0120] The results of the cell ROS inhibition experiment show that after UVA irradiation, the fluorescence intensity in human skin fibroblasts HES increases significantly, and the fluorescence intensity of the active oxygen fluorescent probe (DCFH-DA) increases to 283.92% relative to the blank control group, indicating that the UVA light damage model is successfully established.

[0121] The positive control vitamin C and different concentrations of rice fermentation liquid freeze-dried powder and succinic acid are added in advance, and then subjected to UVA irradiation. The results show that vitamin C has a significant effect on reducing the intracellular fluorescence, and the fluorescence is almost invisible. After adding 10 mg / mL, 5 mg / mL, 2.5 mg / mL rice fermentation liquid freeze-dried powder, the fluorescence intensity is reduced to 11.82%, 18.34%, and 35.32%, respectively. After adding 1 mg / mL, 0.5 mg / mL, and 0.25 mg / mL succinic acid, the fluorescence intensity is reduced to 126.23%, 142.65%, and 169.68%, respectively. It is shown that the rice fermentation liquid freeze-dried powder has strong antioxidant effect; and succinic acid has certain antioxidant effect under certain concentration, and the specific effect intensity needs to be verified by repeated experiments, and the results are shown in Figure 1 、 2 .

[0122] Among them, A: blank control group; B: damage model group; C: vitamin C positive control; D: 10 mg / mL rice freeze-dried powder; E: 5 mg / mL rice freeze-dried powder; F: 2.5 mg / mL rice freeze-dried powder; G: 1 mg / mL succinic acid; H: 0.5 mg / mL succinic acid; I: 0.25 mg / mL succinic acid;

[0123] Figure 2 In the figure, control is normal untreated cells without ROS oxidative damage, while vitamin C has strong antioxidant effect and the intracellular ROS is basically 0.

[0124] Effect Example 6

[0125] In this effect example, the human keratinocytes HaCat in the blank control group, the damage model group, the positive control group and the different sample groups were studied by cell ROS inhibition experiment to verify the repair effect of the rice fermentation liquid freeze-dried powder on the UVB damaged human keratinocytes HaCat.

[0126] The specific operation steps of the cell ROS inhibition experiment are as follows:

[0127] (1) Cell inoculation: human keratinocytes HaCat were inoculated in 24-well culture plates at a cell density of 2-3 x 10 5 per hole, and then placed in a CO2 cell incubator for 24 h.

[0128] (2) According to the experimental grouping, the test substances were added into the cells and cultured for 24 h. The experimental groups were as follows: serum-free medium (blank control group, which was not subjected to UVB irradiation later), serum-free medium (damage model group, which was subjected to UVB irradiation later), vitamin C (200 ug / mL, positive control group), serum-free medium respectively prepared with rice freeze-dried powder (10, 5, 2.5 mg / mL, sample group), succinic acid (1, 0.5, 0.25 mg / mL, sample group).

[0129] (3) Except for the blank control group, the rest of the cells were subjected to UVB irradiation (2 J / cm 2 ).

[0130] (4) Discard the culture solution, wash and fix the cells, and add the working concentration of active oxygen fluorescent probe DCFH-DA reagent into the cells according to the reagent operation instruction for co-incubation.

[0131] (5) After incubation, the cells were photographed under an inverted fluorescence microscope and the fluorescence intensity was counted.

[0132] The results showed that the fluorescence intensity in the cells increased significantly after UVB irradiation, and the DCF fluorescence intensity increased to 263.45% compared with the blank control group, indicating that the UVB light damage model was successfully established; after pre-adding the positive control vitamin C and different concentrations of rice freeze-dried powder and succinic acid and then UVB irradiation, the results showed that vitamin C had a significant effect on reducing intracellular fluorescence, and the fluorescence was almost invisible; after adding 10 mg / mL, 5 mg / mL, 2.5 mg / mL rice freeze-dried powder, the fluorescence intensity decreased to 22.32%, 28.36%, and 33.12%, respectively; and after adding 1 mg / mL, 0.5 mg / mL, and 0.25 mg / mL succinic acid, the fluorescence intensity decreased to 134.13%, 151.35%, and 189.52%, respectively, indicating that the rice freeze-dried powder had strong antioxidant effect, and the succinic acid had certain antioxidant effect under certain concentration conditions.

[0133] According to the above effect examples, it can be seen that the test concentration of choline and succinic acid has a repairing effect on skin damage caused by UVA or UVB irradiation, and the test concentration of rice fermentation broth freeze-dried powder also has a repairing effect on skin damage caused by UVA or UVB irradiation.

[0134] Since the rice fermentation broth freeze-dried powder contains choline and succinic acid at the same time, it is verified that the rice fermentation broth freeze-dried powder has a certain repairing effect on human skin fibroblast HES damage or HaCaT cell damage, and its effective concentration is low, and it has no toxic side effects.

[0135] The above description of the embodiments is for the convenience of the ordinary skilled person in the art to understand and use the invention. Those skilled in the art can easily make various modifications to these embodiments, and apply the general principles described herein to other embodiments without creative labor. Therefore, the present application is not limited to the above embodiments, and the improvements and modifications made by those skilled in the art without departing from the scope of the present application should be within the scope of protection of the present application.

Claims

1. Use of rice ferment filtrate freeze-dried powder for the preparation of a product for repairing skin damage, characterized in that, The rice fermentation broth freeze-dried powder is added to a product for repairing skin damage, and the concentration of the rice fermentation broth freeze-dried powder in the product is 1.25-10 mg / mL. The rice fermentation broth freeze-dried powder is prepared from rice fermentation broth, and the rice fermentation broth freeze-dried powder contains succinic acid and choline. The preparation method of the rice fermentation broth is as follows: (1) rice powder is heated and stirred with water to become rice slurry, and β-glucanase is added to the rice slurry, the enzyme addition amount is 0.001%-0.5% of the mass of the rice slurry, and the enzyme activity is 150-200 U / g, and enzyme hydrolysis is carried out to obtain rice slurry enzyme hydrolyzate; (2) yeast is inoculated into the rice slurry enzyme hydrolyzate, and the yeast used is saccharomyces veronae, and the fermentation conditions are as follows: fermentation temperature 20-40℃, fermentation time 24-48 hours, and pH control during fermentation is 6-6.5, and rice fermentation broth is obtained after fermentation; (3) the rice fermentation broth after fermentation is concentrated by reverse osmosis technology to enrich active substances, and after sterilization by heating and cooling and filtration, the concentrated rice fermentation broth containing active substances is obtained. In the rice fermentation broth obtained after step (2), the content of succinic acid is 1.6-3.2 mg / mL, and the content of choline is 2.1-4.1 mg / mL.

2. Use of the rice fermentation broth freeze-dried powder according to claim 1 in the preparation of a product for repairing skin damage, characterized in that, In the product, the concentration of the rice fermentation broth freeze-dried powder is 5-10 mg / mL.

3. Use of the rice fermentation broth freeze-dried powder according to claim 2 for the preparation of a product for repairing skin damage, characterized in that, The skin damage refers to skin damage caused by UVA or UVB irradiation.

4. Use of the rice fermentation broth freeze-dried powder according to claim 1 in the preparation of a product for repairing skin damage, characterized in that, The skin damage refers to skin damage caused by damage to human keratinocytes HaCat and / or human skin fibroblasts HES caused by ultraviolet rays.

5. Use of the rice fermentation broth freeze-dried powder according to claim 1 in the preparation of a product for repairing skin damage, characterized in that, The product is a product that can reduce ROS generation caused by UV and repair damage to human skin fibroblasts HES or HaCaT cells.

6. Use of the rice fermentation broth freeze-dried powder according to claim 1 in the preparation of a product for repairing skin damage, characterized in that, The product is selected from a pharmaceutical product or a cosmetic product.

7. Use of the freeze-dried powder of rice fermentation broth according to claim 6 for the preparation of a product for repairing skin damage, characterized in that, The product also includes a carrier, excipient or diluent allowed in a pharmaceutical product or a cosmetic product.

8. Use of the rice fermentation broth freeze-dried powder according to claim 1 in the preparation of a product for repairing skin damage, characterized in that, The product is an emulsion, a paste, a capsule or a tablet.

9. Use of the rice fermentation broth freeze-dried powder according to claim 1 in the preparation of a product for repairing skin damage, characterized in that, The product is an oral preparation or an injection.

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