Schizophyllum commune anti-aging composition for regulating cell rhythm and promoting cell autophagy as well as preparation method and application of schizophyllum commune anti-aging composition

Abstract

The invention discloses a schizophyllum commune anti-aging composition capable of regulating cell rhythm and promoting cell autophagy and a preparation method and application of the schizophyllum commune anti-aging composition capable of regulating cell rhythm and promoting cell autophagy, and the schizophyllum commune anti-aging composition capable of regulating cell rhythm and promoting cell autophagy comprises, by weight, 100 parts of schizophyllum commune anti-aging composition capable of regulating cell rhythm and promoting cell autophagy. The composition is prepared from the following components in parts by variety: 0.2 to 2 parts of glutamine amino ethyl imidazole, 0.5 to 20 parts of wheat germ extract and 82 to 95.8 parts of schizophyllum commune fermentation liquor. According to the application, glutamine amino ethyl imidazole and active ingredients in wheat germ and schizophyllum commune fermentation liquor can be effectively compounded; the content of active ingredients such as total sugar and polypeptide in the prepared composition is high. When the product is used, the activity of lysosome and mitochondria in cells can be remarkably improved, the activity of superoxide dismutase is very excellent, and meanwhile, the cell rhythm can be regulated and cell autophagy can be promoted, so that the product prepared by the invention has ideal effects of resisting oxidation, resisting aging, regulating the cell rhythm and promoting cell autophagy; the requirements of modern skin external preparations are met.

Description

Technical Field

[0001] The present application belongs to the technical field of cosmetics, and in particular relates to a Schizophyllum anti-aging composition that regulates cell rhythm and promotes cell autophagy, as well as a preparation method and application thereof. Background Art

[0002] Aging, also known as aging, is an inevitable stage in the process of life activities. Skin aging is the most obvious in the process of body aging. Skin aging not only causes wrinkles, sagging and pigmentation, but also reduces epidermal metabolism, weakens the ability of epidermal repair damage and barrier protection. There are many ingredients with anti-aging effects, but few ingredients can be both efficient and gentle.

[0003] Disordered biological rhythm is one of the reasons for premature skin aging. When the physiological activity of the skin does not adapt to changes in environmental conditions, it may weaken the skin's natural defense ability. Glutamylaminoethylimidazole has a certain effect on regulating skin biological rhythms, but it cannot be used as the main active ingredient because it is irritating and has limited anti-aging effects.

[0004] Wheat germ accounts for about 3% of wheat grains. It contains 15-40% high-quality protein, 10-20% oil and fat, edible fiber, minerals, vitamins and other ingredients that are beneficial to human health. It is praised by nutritionists as "natural nutrition bank for humans". However, the current use of wheat germ is mainly concentrated in three aspects: first, most wheat germ is mixed into bran and used as feed, resulting in a great waste of resources; second, it is directly added to food, such as wheat germ baked food, soup, snacks, etc.; third, wheat germ oil is extracted, and the defatted wheat germ is used as feed and sold cheaply. It can be seen that the rational use of wheat germ has not been achieved at this stage. Moreover, people's research and development of wheat germ remains at the level of nutrient source, and the research on its health function is not in-depth enough, resulting in limited application.

[0005] Schizophyllum is a precious edible fungus. The active ingredients in its fruiting bodies, mycelium and fermentation liquid have pharmacological activities such as anti-tumor, anti-bacterial and anti-inflammatory, anti-radiation, and improving the body's immunity. Its own effective ingredients and fermentation products have good application prospects in the food industry, medicine and cosmetic research. However, relying solely on the active ingredients produced by Schizophyllum to resist skin aging has limited beauty effects.

[0006] Therefore, it is of great significance to develop a product that can effectively utilize glutamylaminoethylimidazole, wheat germ and Schizophyllum fermentation broth to give full play to the synergistic effect of the three and take into account both high-efficiency anti-aging and mild and non-irritating effects. Summary of the invention

[0007] The technical problem to be solved by the present application is to overcome the defects of the prior art such as glutamylaminoethylimidazole being irritating and having limited anti-shaking effect, wheat germ being limited in application and unable to be effectively utilized, and Schizophyllum fermentation broth having limited efficacy, and to provide a Schizophyllum anti-aging composition that regulates cell rhythm and promotes cell autophagy, and a preparation method and application thereof. The Schizophyllum anti-aging composition that regulates cell rhythm and promotes cell autophagy prepared in the present application has ideal antioxidant properties, high safety, and can be widely used in the field of skin topical preparations.

[0008] This application adopts the following technical solutions to solve the above technical problems:

[0009] The present application provides a Schizophyllum anti-aging composition for regulating cell rhythm and promoting cell autophagy, which is composed of the following components in 100 parts by weight: 0.2 to 2 parts of glutamylaminoethylimidazole, 0.5 to 20 parts of wheat germ extract and 83 to 95.8 parts of Schizophyllum fermentation broth.

[0010] In some embodiments, the weight proportion of the glutamylaminoethylimidazole is preferably 0.5 to 1 part, and more preferably 0.2 to 0.5 part.

[0011] In some embodiments, the weight proportion of the wheat germ extract is preferably 4 to 16 parts, more preferably 4 to 8 parts.

[0012] In some embodiments, the weight proportion of the Schizophyllum fermentation broth is preferably 83 to 99 parts, more preferably 91.5 to 95.8 parts.

[0013] In some embodiments, the wheat germ extract contains spermidine.

[0014] In some embodiments, the method for preparing the Schizophyllum fermentation broth comprises the following steps: inoculating the Schizophyllum into the fermentation substrate, fermenting and culturing, and sterilizing to obtain the Schizophyllum fermentation broth.

[0015] Among them, the fermentation substrates include potato starch, glucose, nitrogen source and water.

[0016] Wherein, in the preparation method of the Schizophyllum fermentation broth, the potato starch is 0.5-2 parts, preferably 0.8-1.2 parts. The glucose is 0.1-0.8 parts, preferably 0.2-0.6 parts. The nitrogen source is 0.1-0.8 parts, preferably 0.2-0.6 parts. The water is 95-99 parts, preferably 96-98 parts.

[0017] In the method for preparing the Schizophyllum fermentation broth, the fermentation substrate is sterilized according to conventional procedures in the art before use.

[0018] The sterilization conditions and methods may be conventional conditions and methods for such operations in the art, generally high temperature sterilization.

[0019] When the high temperature sterilization method is used to sterilize the fermentation substrate, the sterilization temperature may be a conventional temperature for such operations in the art, preferably 110-125°C, more preferably 115-121°C.

[0020] When the high temperature sterilization method is used to sterilize the fermentation substrate, the sterilization time can be the conventional time for such operation in the art, preferably 15 to 35 minutes, more preferably 20 to 35 minutes, for example 30 minutes.

[0021] When the high temperature sterilization method is used to sterilize the fermentation substrate, the sterilization pressure may be a conventional pressure for such operations in the art, preferably 0.1-0.14 MPa, more preferably 0.1-0.13 MPa, such as 0.12 MPa.

[0022] Among them, according to the common practice in the art, the sterilization operation may further include a cooling operation, which can generally be cooling to room temperature.

[0023] In the method for preparing the Schizophyllum commune fermentation broth, the Schizophyllum commune is deposited in the General Microbiology Center of the China Culture Collection Administration (CGMCC for short), the address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101, the deposit date is June 5, 2019, and the deposit number is CGMCC No. 17788.

[0024] In the method for preparing the Schizophyllum fermentation broth, the Schizophyllum is prepared according to conventional methods in the art.

[0025] The present application also provides a method for preparing the above-mentioned Schizophyllum anti-aging composition for regulating cell rhythm and promoting cell autophagy, which specifically comprises the following steps: uniformly mixing the glutamyl aminoethyl imidazole, the wheat germ extract and the Schizophyllum fermentation broth to obtain the Schizophyllum anti-aging composition for regulating cell rhythm and promoting cell autophagy.

[0026] In some embodiments, the Schizophyllum sp. is added in the form of a Schizophyllum sp. bacterial liquid.

[0027] Wherein, the concentration of the Schizophyllum in the Schizophyllum bacterial solution is 10 5 ~10 9 CFU / mL, preferably 10 6 ~10 8 CFU / mL.

[0028] In some embodiments, the number of the Schizophyllum bacteria inoculated per unit volume of the water is 5×10 3 ~5×10 7 CFU / mL, preferably, the fermentation culture is carried out on a shaker, and the rotation speed of the shaker is 150-300r / min, more preferably 150-250r / min.

[0029] In some embodiments, the fermentation culture time is 24 to 120 hours, and more preferably 48 to 96 hours.

[0030] In some embodiments, the fermentation culture temperature is 25-35°C, more preferably 25-30°C.

[0031] In some embodiments, the mixing operation may be followed by sterilization and / or adding a preservative according to conventional procedures in the art.

[0032] The sterilization method may be a high temperature sterilization method commonly used in the art.

[0033] When the high temperature sterilization method is used for the sterilization, the sterilization temperature may be a conventional temperature for such operations in the art, preferably 110-125°C, more preferably 115-121°C.

[0034] When the high temperature sterilization method is used for the sterilization, the sterilization time can be the conventional time for such operation in the art, preferably 15 to 35 minutes, more preferably 20 to 30 minutes.

[0035] When the high temperature sterilization method is used for the sterilization, the sterilization pressure may be a conventional pressure for such operations in the art, preferably 0.1 to 0.14 MPa, more preferably 0.1 to 0.13 MPa.

[0036] Wherein, in the process of adding the preservative, the preservative may include p-hydroxyacetophenone and / or 1,2-hexanediol according to the conventional art. When the preservative includes p-hydroxyacetophenone and 1,2-hexanediol, the mass percentage of p-hydroxyacetophenone in the mixed material may be 0.3% to 2%, and the mass percentage of 1,2-hexanediol in the mixed material may be 0.3% to 2%; preferably, the mass percentage of p-hydroxyacetophenone in the mixed material is 0.5%, and the mass percentage of 1,2-hexanediol in the mixed material is 0.5%.

[0037] The process of adding the preservative can be carried out under mixing conditions according to the conventional conditions in the art. The mixing temperature can be the conventional temperature for such operations in the art, preferably 60-80°C, more preferably 70-80°C, for example 75°C.

[0038] The present application also provides a Schizophyllum anti-aging composition for regulating cell rhythm and promoting cell autophagy, which is prepared by the preparation method of the Schizophyllum anti-aging composition for regulating cell rhythm and promoting cell autophagy as described above.

[0039] The present application also provides a use of the above-mentioned Schizophyllum anti-aging composition for regulating cell rhythm and promoting cell autophagy directly as a product, as an additive or as a base in the preparation of a skin topical preparation.

[0040] In some embodiments, the anti-aging agent can be used as at least one of the antioxidant active ingredient, the soothing and anti-allergic active ingredient, and the moisturizing active ingredient in the skin topical preparation.

[0041] The present application also provides a skin external preparation, which includes the Schizophyllum anti-aging composition that regulates cell rhythm and promotes cell autophagy as described above.

[0042] In some embodiments, the skin topical preparation may further include active ingredients conventionally used in the art, generally including any one or more of moisturizing active ingredients, whitening active ingredients, anti-inflammatory active ingredients, anti-allergic active ingredients and antioxidant active ingredients.

[0043] In some embodiments, the external skin preparation comprises a facial mask, an essence or a toner.

[0044] In some embodiments, the mass percentage of the Schizophyllum anti-aging composition that regulates cell rhythm and promotes cell autophagy is 5% to 99% of the skin topical preparation, and more preferably 60% to 99%.

[0045] On the basis of conforming to the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present application.

[0046] The reagents and raw materials used in this application are commercially available.

[0047] The positive and progressive effects of the present application are that the anti-aging composition of Schizophyllum for regulating cell rhythm and promoting cell autophagy prepared by the present application can be effectively compounded with glutamylaminoethylimidazole, as well as active ingredients in wheat germ and Schizophyllum fermentation broth; and the prepared Schizophyllum anti-aging composition for regulating cell rhythm and promoting cell autophagy has a high content of active ingredients such as total sugar and polypeptide. Compared with other compound ingredients, when using this product, it can significantly improve the activity of intracellular lysosomes and mitochondria, and the superoxide activity is very excellent. At the same time, it can also regulate cell autophagy and cell rhythm. It can be seen that the product prepared by the present application has ideal antioxidant, anti-aging and other effects, and meets the functionality and safety requirements of modern skin topicals. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] The present application can be better understood by referring to the description given below in conjunction with the accompanying drawings. The accompanying drawings, together with the following detailed description, are included in this specification and form a part of this specification, and are used to further illustrate the preferred embodiments of the present application and explain the principles and advantages of the present application.

[0049] in:

[0050] Figure 1 It is a comparison chart of the total sugar content of Examples 1 to 3 and Comparative Examples 1 to 6;

[0051] Figure 2 It is a comparison chart of polypeptide contents of Examples 1 to 3 and Comparative Examples 1 to 6;

[0052] Figure 3 This is a comparison diagram of the effects of Examples 1 to 3 and Comparative Examples 1 to 6 on the mitochondrial activity of human immortalized epidermal cells;

[0053] Figure 4 This is a comparison chart of the effects of Examples 1 to 3 and Comparative Examples 1 to 6 on the lysosomal activity of human immortalized epidermal cells;

[0054] Figure 5 This is a comparison chart of the effects of Examples 1 to 3 and Comparative Examples 1 to 6 on the SOD enzyme activity of human immortalized epidermal cells;

[0055] Figure 6 The effects of Examples 1 to 3 and Comparative Examples 1 to 6 on the relative mRNA expression of LC3 in human immortalized epidermal cells;

[0056] Figure 7 The effects of Examples 1 to 3 and Comparative Examples 1 to 6 on the relative mRNA expression of NRF2 in human immortalized epidermal cells;

[0057] Figure 8 The effects of Examples 1 to 3 and Comparative Examples 1 to 6 on the relative expression of HAS mRNA in human immortalized epidermal cells;

[0058] Fig. 9 The effects of Examples 1 to 3 and Comparative Examples 1 to 6 on the relative mRNA expression of COLOCK in human immortalized epidermal cells. DETAILED DESCRIPTION

[0059] The present application is further described below by way of examples, but the present application is not limited to the scope of the examples. The experimental methods without specific conditions in the following examples are selected according to conventional methods and conditions, or according to the product instructions.

[0060] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0061] In the following examples and comparative examples, glutamylaminoethylimidazole is commercially available

[0062] In the following examples and comparative examples, the depository of Schizophyllum commune is the General Microbiological Center of China Microbiological Culture Collection Administration (CGMCC for short), with an address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101, a deposit date of June 5, 2019, and a deposit number of CGMCC No. 17788.

[0063] Example 1

[0064] (1) Preparation process of Schizophyllum fermentation broth: 300 mL of deionized water, 3 g of potato starch, 1.5 g of soybean peptide and 1.5 g of glucose were mixed evenly, sterilized in a high pressure sterilizer at 121°C for 20 min, and cooled to room temperature after sterilization to obtain an initial fermentation system; Schizophyllum was inoculated into the initial fermentation system for fermentation, wherein the number of viable bacteria in the Schizophyllum bacterial broth was 10 7 CFU / mL, the inoculation volume is 15mL [based on the volume of water in the initial fermentation system (300mL)], cultured in a constant temperature oscillator at 28℃ for 48h, and the shaking speed is 180r / min; after the fermentation is completed, the obtained schizophyllum fermentation liquid is sterilized in a high-pressure steam sterilizer at 121℃ for 20min. After the sterilization is completed, the fermentation liquid is cooled to room temperature and centrifuged at a centrifugal speed of 4800r / min for 25min. After the centrifugation, the supernatant is taken and sterilized at a temperature of 121℃ and a pressure of 0.12MPa for 25min. After the sterilization is completed, a preservative needs to be added to the fermentation liquid. During the mixing process with the preservative, the mixing temperature is 75℃; based on the mass of the fermentation stock solution, 0.5% p-hydroxyacetophenone and 0.5% 1,2-hexanediol are added, and the mixture is evenly mixed to obtain the schizophyllum fermentation liquid.

[0065] (2) 1 g of glutamylaminoethylimidazole, 16 g of wheat germ extract and 83 g of Schizophyllum fermentation broth were mixed evenly, and then sterilized in a high pressure sterilizer at 121° C. and 0.12 MPa for 30 min. After sterilization, 0.5 g of 1,2-hexanediol and 0.5 g of p-hydroxyacetophenone were added at 75° C. and mixed evenly to obtain a Schizophyllum anti-aging composition that regulates cell rhythm and promotes cell autophagy.

[0066] Example 2

[0067] Compared with Example 1, the only difference is that the addition amounts of the three substances in step (3) are adjusted to 0.5 g of glutamylaminoethylimidazole, 8 g of wheat germ extract and 91.5 g of Schizophyllum fermentation broth, and the other conditions are the same as those in Example 1.

[0068] Example 3

[0069] Compared with Example 1, the only difference is that the addition amounts of the three substances in step (3) are adjusted to 0.2 g of glutamylaminoethylimidazole, 4 g of wheat germ extract and 95.8 g of Schizophyllum fermentation broth, and the other conditions are the same as those in Example 1.

[0070] Comparative Example 1

[0071] Compared with Example 1, the only difference is that only 100g of Schizophyllum fermentation broth is added, and the specific operations are as follows:

[0072] 100 g of Schizophyllum fermentation broth was mixed evenly, and then sterilized in a high pressure sterilizer at 121° C. and 0.12 MPa for 30 min. After the sterilization, 0.5 g of 1,2-hexanediol and 0.5 g of p-hydroxyacetophenone were added at 75° C. and mixed evenly to obtain a Schizophyllum anti-aging composition that regulates cell rhythm and promotes cell autophagy.

[0073] Comparative Example 2

[0074] Compared with Example 1, the only difference is that the addition amounts of the three substances in step (3) are adjusted to 0.2 g of glutamylaminoethylimidazole, 20 g of wheat germ extract and 79.8 g of Schizophyllum fermentation broth, and the other conditions are the same as those in Example 1.

[0075] Comparative Example 3

[0076] Compared with Example 1, the only difference is that the glutamylaminoethylimidazole in step (3) is replaced by palmitoyl tripeptide-5, and the other conditions are the same as those in Example 1.

[0077] Comparative Example 4

[0078] Compared with Example 1, the only difference is that the wheat germ extract in step (3) is replaced by barley germ extract, and the other conditions are the same as those in Example 1.

[0079] Comparative Example 5

[0080] Compared with Example 3, the only difference is that 3 g of lactobacillus fermentation product is added in step (3), and the other conditions are the same as those in Example 1. The specific operation is as follows:

[0081] 0.2g of glutamylaminoethylimidazole, 4g of wheat germ extract, 92.8g of Schizophyllum fermentation broth and 3g of Lactobacillus fermentation product were mixed evenly, and then sterilized in a high pressure sterilizer at 121°C and 0.12MPa for 30min. After the sterilization, 0.5g of 1,2-hexanediol and 0.5g of p-hydroxyacetophenone were added at 75°C and mixed evenly to obtain a Schizophyllum anti-aging composition that regulates cell rhythm and promotes cell autophagy.

[0082] Comparative Example 6

[0083] Compared with Example 3, the only difference is that the Schizophyllum fermentation broth in step (3) is replaced by a yeast fermentation product. The specific operation is as follows:

[0084] 0.2g of glutamylaminoethylimidazole, 4g of wheat germ extract and 95.8g of yeast fermentation product were mixed evenly, and then sterilized in a high pressure sterilizer at 121°C and 0.12MPa for 30min. After the sterilization, 0.5g of 1,2-hexanediol and 0.5g of p-hydroxyacetophenone were added at 75°C and mixed evenly to obtain a Schizophyllum anti-aging composition that regulates cell rhythm and promotes cell autophagy.

[0085] Effect Example 1 Determination of total sugar content

[0086] The total sugar content of the products prepared in the examples and comparative examples was tested using a total sugar content detection kit with the product number BC2715 produced by Beijing Solebow Technology Co., Ltd. The results are shown in Tables 1 and Figure 1 .

[0087] Table 1

[0088] serial number Total sugar content (mg / mL) Example 1 4.918±0.051 Example 2 4.766±0.046 Example 3 4.824±0.038 Comparative Example 1 3.279±0.004 Comparative Example 2 3.295±0.038 Comparative Example 3 3.152±0.058 Comparative Example 4 2.19±0.026 Comparative Example 5 2.896±0.03 Comparative Example 6 1.977±0.082

[0089] The results show that the total sugar content of the products prepared in Examples 1 to 3 is significantly higher than the total sugar content of the products prepared in Comparative Examples 1 to 6. Figure 1 In the table, ns means no significant difference compared with Example 1, and ***p<0.001 means there is an extremely significant statistical difference compared with Example 1, which is a very significant decrease.)

[0090] Effect Example 2 Determination of polypeptide content

[0091] 1. Reagent preparation

[0092] 1) Reagent A:

[0093] (A) 10g Na 2 CO 3 , 2g NaOH and 0.25g potassium sodium tartrate (KNaC 4 H 4 O 6 ·4H 2 O). Dissolve in 500 ml of deionized water.

[0094] (B) 0.5 g copper sulfate (CuSO 4 ·5H 2 Before each use, mix 50 parts of (A) with 1 part of (B) to obtain reagent A.

[0095] 2) Reagent B: Mix Folin-phenol reagent and deionized water in a ratio of 1:1

[0096] 3) Standard solution: Accurately weigh crystallized bovine serum albumin and dissolve it in deionized water to a concentration of 500 μg / ml.

[0097] 2. Operation method:

[0098] 1) Determination of standard curve: Add 0, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5 ml of standard protein solution (concentration is 500 μg / ml). Make up to 0.5 ml with water, then add 2.5 ml of reagent A to each test tube and place at room temperature for 10 minutes. Add 0.25 ml of reagent B to each tube and mix immediately. Then place at room temperature for 30 minutes, use the first test tube without protein solution as a blank control, and measure the absorbance value of the solution in each test tube at 700 nm. Draw a standard curve with the amount of protein as the horizontal axis and the absorbance value as the vertical axis.

[0099] 2) Sample determination: Take 0.5 ml of sample solution and operate according to the above method. Take 0.5 ml of water instead of sample as blank control. Measure the absorbance value of the solution in each test tube at 700 nm and calculate the polypeptide content through the standard curve. The results are shown in Table 2 and Figure 2 .

[0100] Table 2

[0101] serial number Peptide content (mg / mL) Example 1 1.176±0.01 Example 2 1.178±0.014 Example 3 1.222±0.005 Comparative Example 1 0.558±0.005 Comparative Example 2 0.528±0.005 Comparative Example 3 0.711±0.008 Comparative Example 4 0.693±0.007 Comparative Example 5 0.497±0.01 Comparative Example 6 0.514±0.003

[0102] The results showed that the total sugar content of the products prepared in Examples 1 to 3 was significantly higher than the polypeptide content of the products prepared in Comparative Examples 1 to 6. Figure 2 In the table, ns means no significant difference compared with Example 1, and ***p<0.001 means there is an extremely significant statistical difference compared with Example 1, which is a very significant decrease.)

[0103] Effect Example 3 Mitochondrial Activity Assay

[0104] This experiment uses human immortalized epidermal cells from the Chinese Science Cell Bank to verify the cell mitochondrial activity of the products obtained in the above examples and comparative examples.

[0105] Reagents: 0.25% (containing EDTA) trypsin was produced by GIBCO, USA; DMEM culture medium was produced by GIBCO, USA; double antibody was produced by Corning, USA; CCK-8 was produced by Beijing Biode Biotech Co., Ltd.; fetal bovine serum was produced by GIBCO, USA; phosphate buffer was produced by Beijing Bioregi Biotech Co., Ltd.

[0106] Equipment: WJ-80A-Ⅱ CO 2 The manufacturer of the constant temperature incubator is Shanghai Shengke Instrument Equipment Co., Ltd.; the manufacturer of the Sunrise microplate reader is Tecan Trading Co., Ltd.; the manufacturer of the TL80-2 medical centrifuge is Jiangsu Tianli Medical Equipment Co., Ltd.; the manufacturer of the NUNC 96-well cell culture plate is Thermo Fisher Scientific.

[0107] 1. Experimental steps:

[0108] The products obtained in Examples 1 to 3 and Comparative Examples 1 to 6 were respectively prepared into 1.25% of the test solution in the experimental group using serum-free DMEM medium. Human immortalized epidermal cells were cultured in a medium containing 10% fetal bovine serum and 1% double antibody (1×10 5 The cells were grown in DMEM medium containing 100 U / L penicillin and 100 mg / L streptomycin. 2 In a saturated humidity incubator, when the cell confluence reaches more than 85%, the cells in the logarithmic growth phase are digested with 0.05% trypsin, and the digestion reaction is terminated with serum-containing DMEM. The cell count plate is used to count and the cell suspension concentration is adjusted to 7×10 4 The cell suspension was inoculated into a 96-well plate at a ratio of 100 μL per well and incubated at 37°C and 5% CO 2 Incubate for 12 hours under the same conditions. Remove the old culture medium and wash the cells twice with phosphate buffer. Add 100 μL of the above-prepared sterilized test solution of different concentrations to each well of the experimental group, and make 6 duplicate wells for each test solution; the control group contains cells and serum-free DMEM medium is added; the blank control group has no cells and 100 μL of PBS is added. Then incubate at 37°C and 5% CO 2 Then, 10 μL of CCK-8 solution was added to each well and incubated for another 3 h. The absorbance was measured at 450 nm and the cell survival rate of each group was calculated. The results are shown in Table 3 and Figure 3 .

[0109] The cell viability was calculated as follows:

[0110] Cell survival rate (%) = (A experimental group - A blank control group) / (A control group - A blank control group) × 100%.

[0111] Table 3

[0112]

[0113]

[0114] The results show that after the products prepared in Examples 1 to 3 and Comparative Examples 1 to 6 were used to treat human immortalized epidermal cells, the mitochondrial activity shown in Examples 1 to 3 was significantly higher than that in Comparative Examples 1 to 6. It can be seen that when glutamylaminoethylimidazole, wheat germ extract and Schizophyllum fermentation broth were used in combination, the efficacy was significantly better than that of other products, and Examples 1 to 3 showed an ideal synergistic promotion effect. Figure 3 In the table, ns means no significant difference compared with Example 1, and ***p<0.001 means there is a very significant statistical difference compared with C (blank group), which is a very significant decrease.)

[0115] Effect Example 4 Lysosomal Activity Assay

[0116] This experiment uses human immortalized epidermal cells from the Chinese Science Cell Bank to verify the lysosomal activity of the products obtained in the above examples and comparative examples.

[0117] Reagents: 0.25% (containing EDTA) trypsin was produced by GIBCO, USA; DMEM culture medium was produced by GIBCO, USA; double antibody was produced by Corning, USA; neutrophil proliferation and cytotoxicity detection kit was produced by Shanghai Bio-Tech Biotechnology Co., Ltd.; fetal bovine serum was produced by GIBCO, USA; phosphate buffer was produced by Beijing Bio-Tech Biotechnology Co., Ltd.

[0118] Equipment: WJ-80A-Ⅱ CO 2 The manufacturer of the constant temperature incubator is Shanghai Shengke Instrument Equipment Co., Ltd.; the manufacturer of the Sunrise microplate reader is Tecan Trading Co., Ltd.; the manufacturer of the TL80-2 medical centrifuge is Jiangsu Tianli Medical Equipment Co., Ltd.; the manufacturer of the NUNC 96-well cell culture plate is Thermo Fisher Scientific.

[0119] 1. Experimental steps:

[0120] The products obtained in Examples 1 to 3 and Comparative Examples 1 to 6 were respectively prepared into 1.25% of the test solution in the experimental group using serum-free DMEM medium. Human immortalized epidermal cells were cultured in a medium containing 10% fetal bovine serum and 1% double antibody (1×10 5 The cells were grown in DMEM medium containing 100 U / L penicillin and 100 mg / L streptomycin at 37°C and 5% CO 2 In a saturated humidity incubator, when the cell confluence reaches more than 85%, the cells in the logarithmic growth phase are digested with 0.05% trypsin, and the digestion reaction is terminated with serum-containing DMEM. The cell count plate is used to count and the cell suspension concentration is adjusted to 7×10 4The cell suspension was inoculated into a 96-well plate at a ratio of 100 μL per well and incubated at 37°C and 5% CO 2 Incubate for 12 h under the same conditions, remove the old culture medium, wash the cells twice with phosphate buffer, add 100 μL of the above-prepared sterilized test solution of different concentrations to each well of the experimental group, and make 6 duplicate wells for each test solution; the control group contains cells and serum-free DMEM medium is added; the blank control group has no cells and 100 μL of PBS is added, and then incubate at 37°C, 5% CO 2 Then, 20 μl of neutral red staining solution was added and the cells were incubated in a cell culture incubator for 2 hours. After incubation, the cell culture medium containing neutral red staining solution was removed, the cells were washed twice with PBS, 200 μl of neutral red detection lysis solution was added, and the cells were lysed on a shaker at room temperature for 10 minutes. The absorbance value was measured at a wavelength of 540 nm, and the cell survival rate of each group was calculated. The results are shown in Table 4 and Figure 4 .

[0121] The cell viability was calculated as follows:

[0122] Cell survival rate (%) = (A experimental group - A blank control group) / (A control group - A blank control group) × 100%.

[0123] Table 4

[0124] serial number Lysosomal activity (%) C (blank group) 100 Example 1 100.23±2.38 Example 2 100.91±1.59 Example 3 103.63±2.5 Comparative Example 1 75.28±1.86 Comparative Example 2 74.15±2.95 Comparative Example 3 73.24±2.64 Comparative Example 4 79.59±2.06 Comparative Example 5 77.78±2.34 Comparative Example 6 78.23±1.14

[0125] The results show that after the products prepared in Examples 1 to 3 and Comparative Examples 1 to 6 were used to treat human immortalized epidermal cells, the lysosomal activity shown in Examples 1 to 3 was significantly higher than that in Comparative Examples 1 to 6. It can be seen that when glutamylaminoethylimidazole, wheat germ extract and Schizophyllum fermentation broth were used in combination, the efficacy was significantly better than that of other products, and Examples 1 to 3 showed an ideal synergistic promotion effect. Figure 4 In the table, ns means no significant difference compared with C (blank group), ***p<0.001 means there is an extremely significant statistical difference compared with C (blank group), and it is extremely significantly reduced.)

[0126] Effect Example 5 SOD enzyme (superoxide dismutase) activity determination

[0127] This experiment uses human immortalized epidermal cells from the Chinese Science Cell Bank to verify the lysosomal activity of the products obtained in the above examples and comparative examples.

[0128] Reagents: 0.25% (containing EDTA) trypsin was produced by GIBCO, USA; DMEM culture medium was produced by GIBCO, USA; double antibody was produced by Corning, USA; neutrophil proliferation and cytotoxicity detection kit was produced by Shanghai Bio-Tech Biotechnology Co., Ltd.; fetal bovine serum was produced by GIBCO, USA; phosphate buffer was produced by Beijing Bio-Tech Biotechnology Co., Ltd.

[0129] Equipment: WJ-80A-Ⅱ CO 2 The manufacturer of the constant temperature incubator is Shanghai Shengke Instrument Equipment Co., Ltd.; the manufacturer of the Sunrise microplate reader is Tecan Trading Co., Ltd.; the manufacturer of the TL80-2 medical centrifuge is Jiangsu Tianli Medical Equipment Co., Ltd.; the manufacturer of the NUNC 96-well cell culture plate is Thermo Fisher Scientific.

[0130] 1. Experimental steps:

[0131] The products obtained in Examples 1 to 3 and Comparative Examples 1 to 6 were respectively prepared into 1.25% of the test solution in the experimental group using serum-free DMEM medium. Human immortalized epidermal cells were cultured in a medium containing 10% fetal bovine serum and 1% double antibody (1×10 5 The cells were grown in DMEM medium containing 100 U / L penicillin and 100 mg / L streptomycin. 2 In a saturated humidity incubator, when the cell confluence reaches more than 85%, the cells in the logarithmic growth phase are digested with 0.05% trypsin, and the digestion reaction is terminated with serum-containing DMEM. The cell count plate is used to count and the cell suspension concentration is adjusted to 5×10 5 The cell suspension was inoculated into a 6-well plate at a ratio of 2 mL per well and incubated at 37°C and 5% CO 2 Incubate for 12 hours under the same conditions. Remove the old culture medium and wash the cells twice with phosphate buffer. Add 2 mL of the above-prepared sterilized test solution of different concentrations to each well of the experimental group; add serum-free DMEM medium to the control group containing cells; add 2 mL of PBS to the blank control group without cells. Then incubate at 37°C and 5% CO 2 Incubate for 24 hours under the same conditions. Aspirate the cell culture medium, wash once with pre-cooled PBS, add 100 μL SOD sample preparation solution, blow and lyse the cells thoroughly; centrifuge at 4°C, 12000g for 5 minutes, and take the supernatant as the sample to be tested.

[0132] Prepare the reagents according to the kit instructions.

[0133] Add the sample to be tested and other solutions to the 96-well plate in sequence according to the table below, add the reaction start working solution and mix thoroughly, see Table 5.

[0134] Table 5

[0135] sample Blank control 1 Blank control 2 Samples to be tested 20μL - - SOD Assay Buffer - 20μL 40μL Enzyme working solution 160μL 160μL 160μL Reaction start-up solution 20μL 20μL -

[0136] Incubate at 37°C for 30 minutes, measure absorbance at 450 nm, and calculate SOD enzyme activity. The results are shown in Table 6 and Figure 5 .

[0137] The calculation formula is as follows:

[0138] Inhibition rate (%) = (A blank control 1-A sample) / (A blank control 1-A blank control 2) × 100%

[0139] SOD enzyme activity = inhibition rate / (1-inhibition percentage) U / mL

[0140] Table 6

[0141] serial number SOD enzyme activity (U / mL) C (blank group) 0.86±0.04 M(Model Group) 0.50±0.04 VC (positive control) 0.72±0.03 Example 1 0.78±0.02 Example 2 0.77±0.01 Example 3 0.76±0.02 Comparative Example 1 0.45±0.01 Comparative Example 2 0.18±0.03 Comparative Example 3 0.36±0.01 Comparative Example 4 0.19±0.03 Comparative Example 5 0.32±0.04 Comparative Example 6 0.3±0.01

[0142] The results show that after the products prepared in Examples 1 to 3 and Comparative Examples 1 to 6 were used to treat human immortalized epidermal cells, the SOD enzyme activity shown in Examples 1 to 3 was significantly higher than that in Comparative Examples 1 to 6. It can be seen that when glutamylaminoethylimidazole, wheat germ extract and Schizophyllum fermentation broth were used in combination, the antioxidant effect was significantly better than the combination effect of other products, showing a more excellent synergistic promotion effect. ( Figure 5 In the table, **p<0.01 indicates that there is a significant statistical difference compared with the blank group; ***p<0.001 indicates that there is an extremely significant statistical difference compared with the blank group, and the level of the protein in the protein is extremely significantly reduced; ### p<0.001, indicating that there is an extremely significant statistical difference compared with the model group.)

[0143] Effect Example 6: Relative mRNA expression of LC3, NRF2, HAS and COLOCK

[0144] TriQuick total RNA extraction reagent was used to extract RNA from HaCaT cells according to the instructions. FastQuant cDNA kit was used for cDNA first-strand synthesis reaction. Fast Super qPCR MasterMix and qRT-PCR were used to detect the cDNA obtained by further reverse transcription. The specific primer sequences are shown in Table 7 below, and the test results are shown in Tables 8 to 11 and Figures 6 to 9 .

[0145] Table 7

[0146] qRT-PCR primer sequence list

[0147]

[0148] Table 8

[0149]

[0150]

[0151] From Table 8 and Figure 6 The results show that the relative mRNA expression of LC3 in Examples 1 to 3 is significantly higher than that in Comparative Examples 1 to 6. It can be seen that when glutamylaminoethylimidazole, wheat germ extract and Schizophyllum fermentation broth are used in combination, the effect of promoting cell autophagy is significantly better than the combination effect of other products, and the examples show a more excellent synergistic promotion effect. ( Figure 6 In the table, **p<0.01 indicates that there is a significant statistical difference compared with the blank group; ***p<0.001 indicates that there is an extremely significant statistical difference compared with the blank group, and the level of the protein in the protein is extremely significantly reduced; ### p<0.001, indicating that there is an extremely significant statistical difference compared with the model group.)

[0152] Table 9

[0153]

[0154]

[0155] From Table 9 and Figure 7 The results show that the relative mRNA expression of NRF2 in Examples 1 to 3 is significantly higher than that in Comparative Examples 1 to 6. It can be seen that when glutamylaminoethylimidazole, wheat germ extract and Schizophyllum fermentation broth are used in combination, the anti-aging effect is significantly better than the combination effect of other products, and the examples show a more excellent synergistic promotion effect. Figure 7 In the table, **p<0.01 indicates that there is a significant statistical difference compared with the blank group; ***p<0.001 indicates that there is an extremely significant statistical difference compared with the blank group, and the level of the protein in the protein is extremely significantly reduced; ### p<0.001, indicating that there is an extremely significant statistical difference compared with the model group.)

[0156] Table 10

[0157] serial number Relative expression of HAS mRNA C (blank group) 1 M(Model Group) 0.49±0.09 VC (positive control) 0.67±0.05 Example 1 0.84±0.06 Example 2 0.88±0.07 Example 3 0.9±0.04 Comparative Example 1 0.41±0.15 Comparative Example 2 0.31±0.02 Comparative Example 3 0.23±0.06 Comparative Example 4 0.24±0.02 Comparative Example 5 0.27±0.02 Comparative Example 6 0.32±0.06

[0158] From Table 10 and Figure 8The results show that the relative expression of HAS mRNA in Examples 1 to 3 is significantly higher than that in Comparative Examples 1 to 6. It can be seen that when glutamylaminoethylimidazole, wheat germ extract and Schizophyllum fermentation broth are used in combination, the anti-aging effect is significantly better than the combination effect of other products, and the examples show a more excellent synergistic promotion effect. Figure 8 In the table, **p<0.01 indicates that there is a significant statistical difference compared with the blank group; ***p<0.001 indicates that there is an extremely significant statistical difference compared with the blank group, and the level of the protein in the protein is extremely significantly reduced; ### p<0.001, indicating that there is an extremely significant statistical difference compared with the model group.)

[0159] Table 11

[0160] serial number Relative mRNA expression of COLOCK C (blank group) 1 M(Model Group) 0.40±0.01 VC (positive control) 1±0.05 Example 1 0.79±0.08 Example 2 0.74±0.04 Example 3 0.81±0.06 Comparative Example 1 0.42±0.04 Comparative Example 2 0.39±0.05 Comparative Example 3 0.32±0.04 Comparative Example 4 0.27±0.03 Comparative Example 5 0.3±0.02 Comparative Example 6 0.31±0.03

[0161] From Table 11 and Fig. 9 The results show that the relative mRNA expression of COLOCK in Examples 1 to 3 is significantly higher than that in Comparative Examples 1 to 6. It can be seen that when glutamylaminoethylimidazole, wheat germ extract and Schizophyllum fermentation broth are used in combination, the effect of regulating cell rhythm is significantly better than the combination effect of other products, and the examples show a more excellent synergistic promotion effect. Fig. 9 In the table, *p<0.05 indicates that there is a statistical difference compared with the model group; **p<0.01 indicates that there is a significant statistical difference compared with the model group; ***p<0.001 indicates that there is an extremely significant statistical difference compared with the model group; ### p<0.001 indicates that there is an extremely significant statistical difference compared with the blank control group, and ns indicates that there is no significant difference compared with the model group.)

[0162] Finally, it should be noted that in this application, the terms "comprises", "includes" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or apparatus that includes a series of elements includes not only those elements, but also other elements that are not explicitly listed, or also includes elements that are inherent to such process, method, article or apparatus.

[0163] Although the present application has been disclosed above through the description of the specific embodiments of the present application, it should be understood that those skilled in the art can design various modifications, improvements or equivalents to the present application within the spirit and scope of the attached scheme. These modifications, improvements or equivalents should also be considered to be included in the scope of protection claimed in the present application.

Claims

1. A Schizophyllum anti-aging composition for regulating cell rhythm and promoting cell autophagy, characterized in that: Based on 100 parts by weight, the invention is composed of the following components: 0.2-2 parts of glutamylaminoethylimidazole, 0.5-20 parts of wheat germ extract and 82-95.8 parts of Schizophyllum fermentation liquid.

2. The Schizophyllum anti-aging composition for regulating cell rhythm and promoting cell autophagy according to claim 1, characterized in that: The Schizophyllum anti-aging composition that regulates cell rhythm and promotes cell autophagy meets at least one of the following conditions: The weight proportion of the glutamylaminoethylimidazole is preferably 0.4 to 1 part, more preferably 0.2 to 0.5 part; The weight proportion of the wheat germ extract is preferably 3 to 16 parts, more preferably 4 to 8 parts; The weight proportion of the Schizophyllum fermentation broth is preferably 83 to 99 parts, more preferably 91.5 to 95.8 parts; The wheat germ extract contains spermidine.

3. The Schizophyllum anti-aging composition for regulating cell rhythm and promoting cell autophagy according to claim 1, characterized in that: The method for preparing the Schizophyllum fermentation broth comprises the following steps: inoculating the Schizophyllum into the fermentation substrate, fermenting and culturing, and sterilizing to obtain the Schizophyllum fermentation broth; Preferably, the fermentation substrate comprises potato starch, glucose, a nitrogen source and water.

4. The Schizophyllum anti-aging composition for regulating cell rhythm and promoting cell autophagy according to claim 3, characterized in that: The method for preparing the Schizophyllum fermentation broth satisfies at least one of the following conditions: The potato starch is 0.5 to 2 parts, preferably 0.8 to 1.2 parts; The glucose is 0.1 to 0.8 parts, preferably 0.2 to 0.6 parts; The nitrogen source is 0.1 to 0.8 parts, preferably 0.2 to 0.6 parts; The water is 95-99 parts, preferably 96-98 parts; The fermentation substrate includes a sterilization operation before use. Preferably, the sterilization method is a high-temperature sterilization method. When the high-temperature sterilization method is used to sterilize the fermentation substrate, the sterilization temperature is 110-125°C, and more preferably 115-121°C; when the high-temperature sterilization method is used to sterilize the fermentation substrate, the sterilization time is 15-35min, and more preferably 20-35min, for example 30min; when the high-temperature sterilization method is used to sterilize the fermentation substrate, the sterilization pressure is 0.1-0.14M pa, and more preferably 0.1-0.1 3MPa, for example 0.12MPa.

5. The Schizophyllum anti-aging composition for regulating cell rhythm and promoting cell autophagy according to claim 3, characterized in that: The method for preparing the Schizophyllum fermentation broth satisfies at least one of the following conditions: The Schizophyllum commune is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC), the address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101, the deposit date is June 5, 2019, and the deposit number is CGMCC No. 17788; The schizophyllum is added in the form of a schizophyllum bacterial liquid. Preferably, the concentration of the schizophyllum in the schizophyllum bacterial liquid is 10 5 ~10 9 CFU / mL, preferably 10 6 ~10 8 CFU / mL; The number of the Schizophyllum bacteria inoculated per unit volume of the water is 5×10 3 ~5×10 7 CFU / mL, preferably, the fermentation culture is carried out on a shaker, and the rotation speed of the shaker is 150-300r / min, more preferably 150-250r / min; The fermentation culture time is 24 to 120 hours, preferably 48 to 96 hours; The fermentation temperature is 25-35°C, more preferably 25-30°C; The sterilization method is high-temperature sterilization; when the high-temperature sterilization method is used for sterilization, the sterilization temperature is preferably 110-125°C, and more preferably 115-121°C; when the high-temperature sterilization method is used for sterilization, the sterilization time is preferably 15-35 minutes, and more preferably 20-35 minutes; when the high-temperature sterilization method is used for sterilization, the sterilization pressure is preferably 0.1-0.14MPa, and more preferably 0.1-0.13MPa.

6. The Schizophyllum anti-aging composition for regulating cell rhythm and promoting cell autophagy according to claim 3, characterized in that: After the sterilization operation, the process further includes cooling and / or centrifuging to collect the supernatant; Preferably, the cooling is cooling to room temperature; Preferably, the centrifugal speed is preferably 3000-9000 r / min, more preferably 4000-6000 r / min; Preferably, the radius of the centrifuge is preferably 8 to 15 cm; Preferably, the centrifugation time is preferably 10 to 40 min, more preferably 20 to 30 min.

7. A method for preparing a Schizophyllum anti-aging composition for regulating cell rhythm and promoting cell autophagy as claimed in any one of claims 1 to 6, characterized in that: The method comprises the following steps: uniformly mixing the glutamylaminoethylimidazole, the wheat germ extract and the Schizophyllum fermentation liquid to obtain the Schizophyllum anti-aging composition for regulating cell rhythm and promoting cell autophagy; Preferably, the mixing operation further includes sterilization and / or home preservative operation; More preferably, the sterilization method is a high temperature sterilization method. When the high temperature sterilization method is used to sterilize the fermentation substrate, the sterilization temperature is 110-125°C, and more preferably 115-121°C; when the high temperature sterilization method is used to sterilize the fermentation substrate, the sterilization time is 15-35min, and more preferably 20-35min, for example, 30min; when the high temperature sterilization method is used to sterilize the fermentation substrate, the sterilization pressure may be 0.1-0.14M pa, and more preferably 0.1-0.13MPa, for example, 0.12MPa; More preferably, in the process of adding the preservative, the preservative may include p-hydroxyacetophenone and / or 1,2-hexanediol according to the conventional art; when the preservative includes the p-hydroxyacetophenone and the 1,2-hexanediol, the mass percentage of the p-hydroxyacetophenone in the material obtained after mixing may be 0.3% to 2%, and the mass percentage of the 1,2-hexanediol in the material obtained after mixing may be 0.3% to 2%; preferably, the mass percentage of the p-hydroxyacetophenone in the material obtained after mixing is 0.5%, and the mass percentage of the 1,2-hexanediol in the material obtained after mixing is 0.5%.

8. A Schizophyllum anti-aging composition for regulating cell rhythm and promoting cell autophagy, characterized in that: The composition is prepared by the method for preparing the Schizophyllum anti-aging composition for regulating cell rhythm and promoting cell autophagy as described in claim 7.

9. Use of the Schizophyllum anti-aging composition for regulating cell rhythm and promoting cell autophagy as claimed in any one of claims 1 to 6 and / or the Schizophyllum anti-aging composition for regulating cell rhythm and promoting cell autophagy as claimed in claim 8 directly as a product, as an additive or as a base in the preparation of a skin topical preparation; Preferably, the Schizophyllum anti-aging composition that regulates cell rhythm and promotes cell autophagy is used as at least one of the antioxidant active ingredient, anti-aging active ingredient and moisturizing active ingredient in the skin external preparation; More preferably, the antioxidant active ingredients include antioxidant active ingredients that increase intracellular lysosomal activity and antioxidant active ingredients that promote SOD enzyme activity; More preferably, the anti-aging active ingredient is an anti-aging active ingredient that increases the activity of mitochondria in cells.

10. A skin external preparation, characterized in that: It comprises the Schizophyllum anti-aging composition for regulating cell rhythm and promoting cell autophagy as described in any one of claims 1 to 6 and / or the Schizophyllum anti-aging composition for regulating cell rhythm and promoting cell autophagy as described in claim 8; Preferably, the skin external preparation further comprises any one or more of a moisturizing active ingredient, a whitening active ingredient, an anti-inflammatory active ingredient, an anti-allergic active ingredient and an antioxidant active ingredient; Preferably, the skin external preparation comprises a facial mask, an essence or a toner; Preferably, the mass percentage of the Schizophyllum anti-aging composition that regulates cell rhythm and promotes cell autophagy is 5% to 99% of the skin external preparation, and more preferably 60% to 99%.

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