Epimedium extract for activating autophagy of cells, and preparation method and application thereof

Abstract

The present application relates to a kind of activation of cell autophagy Epimedium extract and its preparation method and application, the present application is prepared using specific fermentation bacteria and composite enzyme Epimedium extract, improve the content of Epimedium in Epimedium extract Epimedium secondary glycoside I, the Epimedium extract can be up-regulated lysosome mannose glycosidase (LManV) expression, significantly enhance cell autophagy flow, promote autophagosome and lysosome fusion, efficiently reverse the p62 / Ref (2) P accumulation caused by autophagy function decline, maintain intracellular environment steady, delay cell aging speed, provide support for organism anti-aging from cell level, help to improve the physical condition related to aging, maintain organism activity, and safe and nontoxic to human body.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to an epimedium extract that activates autophagy, its preparation method, and its application. Background Technology

[0002] Autophagy is a highly conserved intracellular process that involves the degradation and recycling of damaged organelles, misfolded proteins, and pathogens by lysosomes. The integrity of autophagic flux is closely related to cellular homeostasis, aging, and various diseases such as cancer, neurodegenerative diseases, and infections. Autophagic function declines with age and is considered a key driver of aging. Therefore, the search for compounds that can safely and effectively activate autophagic flux is currently a hot topic in anti-aging and related disease drug development.

[0003] Flavonoids are widely found in plants and have been reported to have the potential to activate autophagy. Epimedium extract, a flavonoid derived from the traditional Chinese medicine Epimedium, has been shown in existing studies to possess anti-inflammatory and antioxidant activities. However, whether it can directly regulate and enhance cellular autophagic flux, particularly its molecular targets and mechanisms of action, remains unclear. Clarifying its autophagy regulatory mechanism and function will provide important scientific evidence and application directions for the in-depth development of Epimedium extract in pharmaceuticals and skincare products. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide an epimedium extract that activates autophagy, its preparation method, and its application.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] In a first aspect, the present invention provides a method for preparing an epimedium extract that activates autophagy, comprising the following steps:

[0007] S1. Wash fresh Epimedium, dry it, grind it into powder, and sift it to obtain Epimedium powder;

[0008] S2. Add 10-50 times the weight of water to the Epimedium powder, stir well, and sterilize at high temperature to obtain the fermentation substrate;

[0009] S3. Inoculate the fermentation substrate with a compound bacterial solution for fermentation, and sterilize after the reaction to obtain Epimedium fermentation broth; wherein, the compound bacterial solution is a mixed bacterial solution of Lactobacillus plantarum, Lactobacillus acidophilus and Lactobacillus casei.

[0010] S4. Add a compound enzyme to the Epimedium fermentation broth for enzymatic hydrolysis. After the enzymatic hydrolysis is completed, inactivate the enzyme and spray dry to obtain the Epimedium extract that activates cell autophagy. The compound enzyme is pectin lyase and pullulanase.

[0011] The Epimedium extract of this invention utilizes a specific complex of bacteria (Lactobacillus plantarum, Lactobacillus acidophilus, and Lactobacillus casei) to perform cell wall disruption and pre-conversion of Epimedium through metabolic complementarity, generating easily enzymatically hydrolyzable intermediates. Then, a complex enzyme (pectin lyase and pullulanase) is used to efficiently and directionally convert these precursors into icariin I. This invention employs a sequential and mutually reinforcing process between bacteria and enzymes to form a highly efficient preparation system, yielding an Epimedium extract containing a high content (1-2%) of icariin I.

[0012] Preferably, the viable count of the compound bacterial solution is 1.0 × 10⁻⁶. 9 –5.0×10 9 CFU / mL; the inoculation concentration of the compound bacterial solution is 0.7%-3% v / v, and the viable count ratio of Lactobacillus plantarum, Lactobacillus acidophilus and Lactobacillus casei bacterial solutions is (4-5):(0.2-1):(0.2-1).

[0013] Preferably, the *Lactobacillus plantarum* was purchased from Guangdong Provincial Microbial Culture Collection Center, with accession number GDMCC NO. 64374; the *Lactobacillus acidophilus* was purchased from China General Microbiological Culture Collection Center, with accession number CGMCC 1.1854; and the *Lactobacillus casei* was purchased from Ningbo Taisto Biotechnology Co., Ltd., with catalog number TS277920.

[0014] Preferably, in step S3, the fermentation culture temperature is 25-35℃ and the time is 36-60h.

[0015] Preferably, the pectin lyase has an enzyme activity of 5000-20000 U / g and is added at a rate of 0.15-1.5% of the weight of the Epimedium fermentation broth; the pullulanase has an enzyme activity of 1000-8000 U / g and is added at a rate of 0.05-0.5% of the weight of the Epimedium fermentation broth; the enzymatic hydrolysis temperature is 40-60℃ and the time is 1-3h.

[0016] Preferably, the enzyme inactivation temperature in step S4 is 90-100℃ and the time is 20-30 min.

[0017] In a second aspect, the present invention provides an epimedium extract that activates autophagy, prepared by the method for preparing the epimedium extract that activates autophagy described in the first aspect.

[0018] Thirdly, the present invention provides the application of the Epimedium extract that activates autophagy as described in the second aspect in the preparation of pharmaceuticals and skin care products with autophagy-activating effects.

[0019] The skincare products described herein can improve skin condition, including delaying skin aging, repairing the skin barrier, or improving skin elasticity. The medication described herein can be used to treat diseases related to impaired autophagy (such as aging, intestinal diseases, and neurodegenerative diseases).

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0021] (1) This invention solves the problems of low activity and uneven composition of traditional Epimedium extract by using the bacterial-enzyme sequential synergistic technology. Epimedium extract is prepared by using specific fermentation bacteria and compound enzymes, which increases the content of epimedium glycoside I in Epimedium extract. This Epimedium extract can significantly enhance cell autophagy flux by upregulating the expression of lysosomal mannosidase (LManV), promote the fusion of autophagosomes and lysosomes, efficiently reverse the accumulation of p62 / Ref(2)P caused by the decline of autophagy function, maintain the homeostasis of intracellular environment, delay the rate of cell aging, and provide support for anti-aging of the body at the cellular level. Human experiments have confirmed that it can help improve the physical condition related to aging and maintain the vitality of the body.

[0022] (2) The Epimedium extract prepared by the present invention has a high content of Epimedium glycoside I by means of the preparation technology of bacterial enzyme synergy. Its core health value lies in enhancing the autophagy function of cells through a clear molecular mechanism, thereby achieving the core goal of anti-aging. At the same time, it can help maintain intestinal health, nourish nerve function, and optimize the overall metabolism and vitality of the body. It has broad application prospects in the field of health care, and is especially suitable for developing anti-aging and intestinal care health care products.

[0023] (3) Based on its autophagy-promoting mechanism, Epimedium extract has great potential in developing drugs to treat diseases related to low autophagy function (such as aging, intestinal diseases, and neurodegenerative diseases). Attached Figure Description

[0024] Figure 1 Hierarchical clustering analysis diagram of differentially expressed genes between the elderly control group and the intervention group of Example 1;

[0025] Figure 2 The image shows the results of screening and analysis of the intestinal LManV gene in 30-day-old fruit flies fed according to Examples 1-3 and Comparative Examples 1-7. Detailed Implementation

[0026] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0027] The raw materials used in the following examples and comparative examples are from the following sources:

[0028] Lactobacillus plantarum 1: purchased from Guangdong Provincial Microbial Culture Collection Center, accession number GDMCC NO.64374;

[0029] Lactobacillus plantarum 2: purchased from China General Microbiological Culture Collection Center, accession number CGMCC1.119;

[0030] Lactobacillus acidophilus: purchased from China General Microbiological Culture Collection Center, accession number CGMCC 1.1854;

[0031] Lactobacillus casei: purchased from Ningbo Taisto Biotechnology Co., Ltd., product number TS277920;

[0032] Pectin lyase: Purchased from Shandong Pingju Biotechnology Co., Ltd., product number CAS No. 9015-75-2

[0033] Pullulanase: Purchased from Zhengzhou Givaudan Chemical Products Co., Ltd., product number CAS No. 9075-68-7

[0034] Unless otherwise specified, all other materials and reagents used in the examples are commercially available. Example 1

[0035] A method for preparing an epimedium extract that activates autophagy includes the following steps:

[0036] S1. Wash fresh Epimedium, dry it, grind it into powder, and pass it through a 20-mesh sieve to obtain Epimedium powder;

[0037] S2. Take 100g of Epimedium powder and add it to 1000g of water. Heat and extract at 80℃ for 2 hours. After cooling, obtain the fermentation substrate.

[0038] S3. Take 15 mL of the compound bacterial solution and inoculate it into 1000 mL of fermentation substrate to obtain the fermentation system. Ferment the system in a shaker at 30℃ for 48 h. After fermentation, sterilize at 100℃ for 30 min to obtain the Epimedium fermentation broth; wherein, the total viable count of the compound bacterial solution is 2.5 × 10⁻⁶. 9 CFU / mL, the compound bacterial solution includes Lactobacillus plantarum 1 bacterial solution, Lactobacillus acidophilus bacterial solution and Lactobacillus casei bacterial solution with a live bacteria ratio of 4:1:1;

[0039] S4. Add 1% (w / w) of a complex enzyme (0.75% pectin lyase + 0.25% pullulanase) to the Epimedium fermentation broth, enzymatically hydrolyze at 50℃ for 2 hours, inactivate the enzyme at 100℃ for 20 minutes, filter, discard the precipitate, collect the supernatant, and spray dry to obtain the Epimedium extract that activates autophagy; wherein the activity of the pectin lyase is 10000 U / g, and the activity of the pullulanase is 5000 U / g. Example 2

[0040] A method for preparing an epimedium extract that activates autophagy includes the following steps:

[0041] S1. Wash fresh Epimedium, dry it, grind it into powder, and pass it through a 20-mesh sieve to obtain Epimedium powder;

[0042] S2. Take 50g of Epimedium powder and add it to 1000g of water. Heat and extract at 70℃ for 3 hours. After cooling, obtain the fermentation substrate.

[0043] S3. Take 30 mL of the compound bacterial solution and inoculate it into 1000 mL of fermentation substrate to obtain the fermentation system. Ferment the system in a shaker at 25℃ for 60 h. After fermentation, sterilize at 100℃ for 30 min to obtain the Epimedium fermentation broth; wherein, the total viable count of the compound bacterial solution is 1.0 × 10⁻⁶. 9 CFU / mL, the compound bacterial solution includes Lactobacillus plantarum 1 bacterial solution, Lactobacillus acidophilus bacterial solution and Lactobacillus casei bacterial solution with a live bacteria ratio of 4:1:1;

[0044] S4. Add 2% (w / w) of compound enzyme (1.5% pectin lyase + 0.5% pullulanase) to the Epimedium fermentation broth, enzymatically hydrolyze at 40℃ for 3 hours, inactivate the enzyme at 100℃ for 20 minutes, filter, discard the precipitate, collect the supernatant, and spray dry to obtain the Epimedium extract that activates autophagy; wherein the activity of pectin lyase is 5000 U / g, and the activity of pullulanase is 1000 U / g. Example 3

[0045] A method for preparing an epimedium extract that activates autophagy includes the following steps:

[0046] S1. Wash fresh Epimedium, dry it, grind it into powder, and pass it through a 20-mesh sieve to obtain Epimedium powder;

[0047] S2. Take 20g of Epimedium powder and add it to 1000g of water. Heat at 90℃ for 1 hour and cool to obtain the fermentation substrate.

[0048] S3. Take 7 mL of the compound bacterial solution and inoculate it into 1000 mL of fermentation substrate to obtain the fermentation system. Ferment the system in a shaker at 35℃ for 36 h. After fermentation, sterilize at 100℃ for 30 min to obtain the Epimedium fermentation broth; wherein, the total viable count of the compound bacterial solution is 5.0 × 10⁻⁶. 9 CFU / mL, the compound bacterial solution includes Lactobacillus plantarum 1 bacterial solution, Lactobacillus acidophilus bacterial solution and Lactobacillus casei bacterial solution with a live bacteria ratio of 5:1:1;

[0049] S4. Add 0.2% (w / w) of a complex enzyme (0.15% pectin lyase + 0.05% pullulanase) to the Epimedium fermentation broth, enzymatically hydrolyze at 60℃ for 1 h, inactivate the enzyme at 90℃ for 30 min, filter, discard the precipitate, collect the supernatant, and spray dry to obtain the Epimedium extract that activates autophagy; wherein the activity of the pectin lyase is 20000 U / g, and the activity of the pullulanase is 8000 U / g.

[0050] Comparative Example 1

[0051] The only difference between Comparative Example 1 and Example 1 is that Comparative Example 1 is an epimedium extract that has not undergone fermentation and enzymatic hydrolysis. Its preparation method includes the following steps: washing, drying, and grinding fresh epimedium into powder, which is then passed through a 20-mesh sieve to obtain epimedium powder. 100g of epimedium powder is added to 1000g of water, and the mixture is heated at 80℃ for 2 hours. After cooling, it is spray-dried to obtain the epimedium extract.

[0052] Comparative Example 2

[0053] The only difference between Comparative Example 2 and Example 1 is that Comparative Example 2 is an Epimedium extract that has only undergone fermentation with a compound bacteria and has not undergone enzymatic hydrolysis. The specific preparation method is as follows:

[0054] S1. Wash fresh Epimedium, dry it, grind it into powder, and pass it through a 20-mesh sieve to obtain Epimedium powder;

[0055] S2. Take 100g of Epimedium powder and add it to 1000g of water. Heat it at 80℃ for 2 hours to sterilize. After cooling, you will get the fermentation substrate.

[0056] S3. Inoculate 15 mL of the compound bacterial solution into 1000 mL of fermentation substrate to obtain a fermentation system. Ferment the system in a shaker at 30 °C for 48 h. After fermentation, sterilize at 100 °C for 30 min to obtain the Epimedium fermentation broth. Filter, discard the precipitate, collect the supernatant, and spray-dry to obtain the Epimedium extract. The total viable count of the compound bacterial solution is 2.5 × 10⁻⁶. 9 CFU / mL, the compound bacterial solution includes Lactobacillus plantarum 1 bacterial solution, Lactobacillus acidophilus bacterial solution and Lactobacillus casei bacterial solution with a live bacteria ratio of 4:1:1.

[0057] Comparative Example 3

[0058] The only difference between Comparative Example 3 and Example 1 is that Comparative Example 3 is an Epimedium extract that has only undergone enzymatic hydrolysis with a compound enzyme and has not undergone fermentation. The specific preparation method is as follows:

[0059] S1. Wash fresh Epimedium, dry it, grind it into powder, and pass it through a 20-mesh sieve to obtain Epimedium powder;

[0060] S2. Take 100g of Epimedium powder and add it to 1000g of water. Heat and sterilize at 80℃ for 2 hours. After cooling, the enzymatic hydrolysate is obtained.

[0061] S3. Add 1% (w / w) of a complex enzyme (0.75% pectin lyase + 0.25% pullulanase) to the enzymatic hydrolysis substrate, hydrolyze at 50℃ for 2 hours, inactivate the enzyme at 100℃ for 20 minutes, filter, discard the precipitate, collect the supernatant, and spray dry to obtain the Epimedium extract.

[0062] Comparative Example 4

[0063] The only difference between Comparative Example 4 and Example 1 is that in the preparation step S3 of Comparative Example 4, Lactobacillus plantarum 1 is replaced with Lactobacillus plantarum 2.

[0064] Comparative Example 5

[0065] The only difference between Comparative Example 5 and Example 1 is that Comparative Example 5 does not add Lactobacillus plantarum 1 bacterial solution to the compound bacterial solution, but uses Lactobacillus acidophilus bacterial solution and Lactobacillus casei bacterial solution with a live bacteria ratio of 1:1 to make up the missing amount.

[0066] Comparative Example 6

[0067] The only difference between Comparative Example 6 and Example 1 is that: Comparative Example 6 does not add Lactobacillus acidophilus culture to the compound bacterial culture, but uses Lactobacillus plantarum 1 culture and Lactobacillus casei culture with a live bacteria ratio of 4:1 to make up the missing amount.

[0068] Comparative Example 7

[0069] The only difference between Comparative Example 7 and Example 1 is that: Comparative Example 7 does not add Lactobacillus casei culture to the compound bacterial culture, but uses Lactobacillus plantarum 1 culture and Lactobacillus acidophilus culture with a live bacteria ratio of 4:1 to make up the missing amount.

[0070] Performance testing

[0071] (I) Detection of Epimedium I content in Epimedium extracts of each group

[0072] The content of epimedium glycoside I in the epimedium extracts of Examples 1-3 and Comparative Examples 1-7 was determined by high performance liquid chromatography (HPLC). Chromatographic analysis was performed using a C18 reversed-phase column with gradient elution using acetonitrile-0.1% phosphoric acid aqueous solution as the mobile phase, and the detection wavelength was 270 nm.

[0073] Accurately weigh icariin I reference standard, dissolve and dilute it with methanol to prepare reference solutions of a series of concentrations. Take about 0.1 g of sample powder (Epimedium extracts of Examples 1-3 and Comparative Examples 1-7), accurately add 25 mL of methanol, sonicate for 30 minutes, and filter to obtain the test solution.

[0074] First, a system suitability test was conducted to confirm that the chromatographic system met the requirements. Then, a series of reference solutions were injected sequentially, and a standard curve was plotted by performing linear regression of peak area against concentration. Finally, the test solution was injected, the chromatogram was recorded, and the content of icariin I in the sample was calculated based on the standard curve.

[0075] The content calculation formula is: Content (mg / g) = (C × V) / W, where C is the concentration of the test solution (μg / mL), V is the solution volume (mL), and W is the sample weight (g). Specific data are shown in Table 1.

[0076] Table 1. Content of Epimedium glycoside I in Epimedium extracts of each group

[0077]

[0078] As shown in Table 1, and combining the data from Example 1 and Comparative Examples 1-3, the content of icariin I in Comparative Example 1 was the lowest among all groups. The content of icariin I in Comparative Examples 2-3 was also significantly lower than that in Example 1. This indicates that enzymatic hydrolysis and fermentation are important steps affecting the precipitation of icariin I from Epimedium extract. If enzymatic hydrolysis and / or fermentation are lacking, the content of icariin I will be reduced.

[0079] Based on the data from Example 1 and Comparative Examples 4-7, it can be seen that the content of icariin I in Comparative Examples 4-7 is also lower than that in Example 1. This indicates that the synergistic fermentation of three bacteria, Lactobacillus plantarum 1, Lactobacillus acidophilus and Lactobacillus casei, can increase the content of icariin I in the extract.

[0080] (II) Activation effect of Epimedium extracts on autophagic flux in human intestinal epithelial cells

[0081] 1. Cell culture and treatment:

[0082] Preparation of sample solution: Add the Epimedium extracts of Examples 1-3 and Comparative Examples 1-7 to water and dilute to a sample solution with a mass concentration of 2 mg / mL;

[0083] Human colon adenocarcinoma cells (Caco-2) were cultured in DMEM complete medium (containing 10% fetal bovine serum and 1% penicillin antibody) at 37°C and 5% CO2.

[0084] A Caco-2 cell senescence model was constructed using doxorubicin. Experimental groups were set up: a control group (Control), a doxorubicin-activated senescence model group (DOXO), and a sample intervention group (DOXO + sample). The Control group used complete culture medium, the DOXO group used complete culture medium containing 250 nM doxorubicin, and the DOXO + sample group used complete culture medium containing 250 nM doxorubicin and 2 mg / mL of sample. Once the cell density reached 70%, the cells were treated according to their respective experimental groups for 24 hours.

[0085] 2. CCK-8 cell proliferation experiment

[0086] The effect of the drug on cell viability was determined using a cell proliferation-toxicity assay kit (CCK-8). First, Caco-2 cells (5 × 10⁻⁶) were... 3 Cells (per well) were seeded in 96-well plates, with 100 μL of complete culture medium added to each well. The plates were incubated at 37°C with 5% CO2 for 12 hours. When the cell density reached 70%, the cells were subjected to drug treatment. Complete culture medium containing different concentrations of Epimedium extract (0, 0.1, 0.2, 0.5, 1, and 2 mg / mL) was added to each well, and the cells were treated for 24 hours. After drug treatment, the old culture medium was discarded, and 100 μL of medium containing 10% CCK-8 reagent was added to each well. The plates were incubated at 37°C in the dark for 2 hours. The absorbance at 450 nm was measured using a microplate reader. Cytotoxicity was calculated using the following formula:

[0087] Cell viability = (absorbance of experimental group - absorbance of blank control) / (absorbance of control group - absorbance of blank control) × 100%; where, if cell viability > 90%, it is judged as non-toxic, and if 90% > cell viability > 80%, it is judged as slightly toxic. The results of the Caco2 cytotoxicity experiment are shown in Table 2.

[0088] Table 2. Results of the cytotoxicity experiment of Epimedium extracts on Caco2 cells in each group.

[0089]

[0090] As shown in Table 2, Comparative Example 1 exhibited slight toxicity to Caco 2 cells at 4 mg / mL, while other examples and comparative examples showed no toxicity within the range of 2 mg / mL. This indicates that fermentation or enzymatic hydrolysis can improve the biosafety of Epimedium.

[0091] 3. Cyto-ID and LysoTracker staining of Caco-2 cells

[0092] Preparation of sample solution: Add the Epimedium extracts of Examples 1-3 and Comparative Examples 1-7 to water and dilute to a sample solution with a mass concentration of 2 mg / mL;

[0093] Caco-2 cells were seeded in 24-well plates with cell spreaders at a density of 8 × 10³ cells / well. Once the cell density reached 70%, the old culture medium was carefully discarded. Control, doxorubicin-activated senescence model (DOXO), sample intervention (DOXO + sample), and chloroquine intervention (DOXO + sample + CQ) were established. The chloroquine intervention group used complete culture medium containing 250 nM doxorubicin, 2 mg / mL sample, and 60 μM chloroquine. After treatment with the drug for 24 hours, the cells were washed twice with buffer containing 5% FBS. At 37°C in the dark, the cells were stained with Cyto-ID (1:1000) for 30 minutes, then the staining solution was replaced with a compound staining solution containing LysoTracker (1:20000) and Hoechst 33342 (1 mg / ml, 1:1000), and incubated for 3 minutes in the dark. The cells were then washed with buffer. Cells were fixed with 4% paraformaldehyde for 20 minutes and washed three times with buffer. Add anti-fluorescence quencher and mount the slide. Observation and image acquisition were performed using a Nikon A1 laser confocal microscope. Experimental results are shown in Table 3.

[0094] 4. Western blot assay

[0095] Preparation of sample solution: Add the Epimedium extracts of Examples 1-3 and Comparative Examples 1-7 to water and dilute to a sample solution with a mass concentration of 2 mg / mL;

[0096] Caco-2 cells were seeded in 6-well plates at a density of 2 × 10⁻⁶ cells / well. 5 / well. Once the cell density reaches 70%, carefully discard the old culture medium. Set up a control group, an doxorubicin-activated senescence model group (DOXO), a sample intervention group (DOXO + sample), and a chloroquine intervention group (DOXO + sample + CQ). In these groups, the doxorubicin concentration was 250 nM; the sample concentration was 2 mg / mL; and the chloroquine concentration was 60 μM. After treating cells with the drugs for 24 hours according to the experimental settings, protein extraction was performed. Pre-chill the centrifuge column and collection tube on ice. Wash cells with pre-chilled PBS, discard the supernatant, add column protein extraction reagent containing protease inhibitors, transfer the lysed cells to the pre-chilled centrifuge column tube, and centrifuge (12000 rpm, 30 seconds). Immediately place the collection tube on ice, discard the centrifuge column, and transfer the protein solution in the collection tube to an EP tube. The primary antibodies used in the subsequent immunoblotting experiments were p62 (1:1000) and β-actin (1:50000). The experimental results are shown in Table 3.

[0097] Table 3 Results of Cyto-ID and LysoTracker staining and Western blot experiments in Caco-2 cells

[0098]

[0099] This invention further utilizes a co-staining strategy of Cyto-ID and Lysotracker dyes combined with the intervention of the lysosomal inhibitor chloroquine to systematically evaluate the effect of samples on autophagic flux in an doxorubicin-activated Caco-2 cell senescence model. Chloroquine blocks autophagy by increasing the pH of lysosomes, inactivating hydrolytic enzymes, inhibiting lysosomal function, and suppressing the fusion of autophagosomes with lysosomes, thus leading to the accumulation of autophagosomes within the cells. Table 3 shows that quantitative analysis using immunofluorescence staining revealed a decrease in autophagic flux in the senescence model group compared to the control group. All examples and comparative examples showed enhanced autophagic flux, with the examples demonstrating significantly better effects than the comparative examples, and the effect was directly proportional to the content of icariin I. Chloroquine treatment caused an increase in lysosomal pH, completely blocking the enhanced autophagic flux effect of the activated samples, resulting in the accumulation of a large number of undegraded autophagosomes.

[0100] During autophagy dynamics, the selective autophagy adaptor protein p62 (a Drosophila Ref(2)P homolog) is specifically recruited to autophagosomes via the LC3 interaction region and degraded in an acidic environment after autophagosome-lysosome fusion. Significant accumulation of p62 occurs when autophagic flux is inhibited. Western blotting showed that p62 protein levels were significantly higher in the doxorubicin-activated aging model than in the control group, while all examples and comparative treatments reduced it. The effects of the examples were significantly better than those of the comparatives, and the effect was directly proportional to the content of icariin I. This effect could be reversed by the lysosomal inhibitor chloroquine.

[0101] (III) Fruit fly experiment

[0102] Fruit flies were reared in a constant temperature and humidity incubator (temperature 25℃, humidity 60%, light period from 7 am to 7 pm). To control the growth density, approximately 200 fertilized eggs were placed in each 150 mL standard culture bottle. Three-day-old adults after emergence were anesthetized using a CO2 anesthesia system, and male and female fruit flies were separated and reared separately.

[0103] To prepare a corn-brown sugar culture medium suitable for Drosophila culture (using 500 mL as an example), accurately weigh 50 g corn flour, 3.2 g dry yeast, 32.7 g brown sugar, and 3.2 g agar powder into a sterile beaker, and add 600 mL of ultrapure water. Heat to boiling four times using a microwave gradient heating method (30 seconds per heating cycle, with intermittent stirring), then cool to room temperature. Next, add 3.5 mL propionic acid and 3.5 mL of 25% methylparaben solution (using 95% ethanol as a solvent) to the culture medium, mix thoroughly, and dispense into sterile Erlenmeyer flasks for subsequent experiments.

[0104] Experimental groups: young control group (5d), senescent control group (30d), and sample intervention group (30d + sample). Three-day-old adults were reared separately in culture media containing different samples (including the examples and comparative examples, sample mass concentration of 1%) (n=50-60 insects / bottle, 4 biological replicates).

[0105] 1. Cyto-ID and LysoTracker co-staining

[0106] Cyto-ID staining selectively labels autophagic vacuoles, while LysoTracker dye accumulates in lysosomes. The combination of the two allows for a better assessment of the entire autophagy process.

[0107] (1) Tissue processing: Dissect the midgut of Drosophila in pre-cooled PBS buffer and immediately perform live staining.

[0108] (2) Cyto-ID staining: Immerse in Cyto-ID working solution (1:1000) under light-protected conditions and incubate at 37°C with shaking for 30 minutes.

[0109] (3) Lysosomal labeling: Replace with a compound staining solution containing LysoTracker (1:20000) and Hoechst 33342 (1 mg / ml, 1:1000) and incubate in the dark for 3 minutes.

[0110] (4) Sample washing: Use PBS buffer gradient centrifugation to wash 3 times (500 rpm, 5 minutes / time).

[0111] (5) Mounting and imaging: Add anti-fluorescence quenching agent to a clean glass slide and mount it. Observation and image acquisition were performed using a Nikon A1 laser confocal microscope. The experimental results are shown in Table 5.

[0112] 2. Western blot

[0113] (1) Protein sample preparation: Pre-cool the centrifuge column and collection tube sleeve on ice. Place 40 fruit flies on the centrifuge column and grind them 60 times with a grinding rod. Add 200 uL of pre-cooled lysis buffer containing protease inhibitors and grind for another 60 times. Centrifuge at 4°C (12000 rpm, 1 minute). Discard the centrifuge column and transfer the protein solution in the collection tube to a new EP tube.

[0114] (2) Protein quantification: The BCA protein quantification kit was used to detect the absorbance at 562 nm using an ELISA reader. The concentration was calculated based on the standard curve, and the protein was standardized to the same concentration based on the detected concentration. The protein was mixed with loading buffer and denatured at 100°C for 10 minutes.

[0115] (3) Electrophoresis and transfer: Load 20 μg of protein into 10% SDS-PAGE. Electrophoresis conditions: stacking gel 80 V, separating gel 120 V. Transfer the protein to a PVDF membrane by wet electrophoresis under the following conditions: 200 mA for 90 minutes.

[0116] (4) Antibody incubation and development: Block with rapid blocking solution for 15 minutes, then incubate overnight at 4°C with primary antibody (Ref(2)P, 1:1000; β-actin, 1:50000). Wash three times with TBST for 10 minutes each time. Incubate at room temperature for 1 hour with goat anti-rabbit secondary antibody or goat anti-mouse secondary antibody. Wash three times with TBST for 10 minutes each time. Perform chemiluminescent reaction with chemiluminescent reagent, and perform development and analysis using the ChemiDoc imaging system. The experimental results are shown in Table 4.

[0117] Table 4 Results of Cyto-ID and LysoTracker staining and Western blot experiments in Drosophila

[0118]

[0119] Co-localization staining analysis using Cyto-ID and Lysotracker fluorescent dyes can assess the dynamic changes in autophagic flux. Cyto-ID dye selectively labels autophagosomes, while Lysotracker dye specifically labels autolysosomes. A significant increase in the ratio of autolysosomes to autophagosomes serves as a biological indicator of enhanced autophagic flux. Combined with the immunofluorescence staining and quantitative results in Table 5, it is evident that autophagic flux was reduced in the aged control group. Both the example and comparative studies significantly promoted autophagosome production and autolysosome formation in the intestine, with the example studies showing significantly better results than the comparative studies. The effect was directly proportional to the content of icariin-1. An increase in the ratio of autolysosomes to autophagosomes significantly enhanced autophagic flux.

[0120] Ref(2)P, as a key cargo receptor for selective autophagy, exhibits a degradation rate exceeding its synthesis rate under sustained autophagy activation, leading to a significant decrease in its homeostatic level. Therefore, its protein expression level can serve as a functional biomarker for autophagic flux. Immunoblotting data showed that compared to the 5-day-old control group, the Ref(2)P protein expression level in aged control flies was significantly upregulated (P<0.05), indicating a decline in basal autophagy activity with aging. After intervention in the examples and comparative examples, the Ref(2)P protein level in aged flies was lower than that in the age-matched control group, demonstrating that the effects of the examples were significantly superior to those of the comparative examples. The effect was directly proportional to the content of icariin-1. This indicates that the examples can more effectively activate the autophagy pathway and enhance autophagic flux.

[0121] 3. Screening and analysis of differentially expressed genes

[0122] Three 30-day-old Drosophila (30d) and three 30-day-old Drosophila treated in Example 1 (30d + Example 1) were selected as experimental materials. Three samples were collected from each group for total RNA extraction. The concentration and purity of the extracted RNA were detected using Nanodrop 2000, RNA integrity was detected by agarose gel electrophoresis, and the RQN value was determined using an Agilent 5300. The detection criteria were: sample concentration ≥30 ng / μL, no pigment contamination, no obvious protein, sugar, or other impurities, RQN ≥4.5, 28 / 23S brightness comparable to 18 / 16S, and sample quality indicators meeting the requirements for library construction and sequencing. Eukaryotic mRNA has a polyA tail at its 3' end. Using magnetic beads with Oligo(dT) to perform AT base pairing with polyA, mRNA can be isolated from total RNA for transcriptome analysis. Next-generation high-throughput sequencing platforms are designed for sequencing short sequence fragments. The enriched mRNA is a complete RNA sequence with an average length of several kb, thus requiring random fragmentation. By adding fragmentation buffer and selecting appropriate conditions, the mRNA can be randomly fragmented into small fragments of approximately 300 bp. Using reverse transcriptase and random primers, one-stranded cDNA is synthesized from the mRNA using reverse transcriptase as a template. This is followed by two-strand synthesis to form a stable double-stranded structure. The double-stranded cDNA has sticky ends, which are padded with End Repair Mix to create blunt ends. An A base is then added to the 3' end to facilitate the subsequent addition of the adapter sequence. The product after adapter ligation is purified and fragment sorted. The sorted product is then used for PCR amplification, and the final purified library is obtained.

[0123] Differential expression analysis between treatment groups was performed using DESeq2, with a selection threshold of |log2FC| ≥ 1 and p-value < 0.05. Enrichment analysis of differentially expressed genes for functional annotation was conducted using the Meiji Biotechnology platform.

[0124] To verify the accuracy of the transcriptome sequencing results, qPCR was used to verify whether the LManV gene level in the gut of 30-day-old Drosophila fed with different samples was upregulated. The LManV transcription level increased after intervention in both the example and comparative cases.

[0125] Experimental Results: Volcano plots visually illustrated the differentially expressed gene sets between the elderly control group and the intervention group of Example 1. A total of 104 significantly differentially expressed genes were found between the two groups (P<0.05), including 49 upregulated genes and 55 downregulated genes. Furthermore, hierarchical cluster analysis was performed on the screened differentially expressed genes, clustering genes with similar or identical expression levels. The results are shown below. Figure 1As shown, anti-aging research has revealed some conserved longevity-promoting metabolic processes, including carbohydrate metabolism. Based on GO, KEGG, EggNOG functional classifications and GSEA, proteins involved in carbohydrate metabolism were specifically enriched, particularly mannosidase activity and lysosomal pathway upregulation. Combined with differential gene screening results, the lysosomal mannosidase V (LManV) gene was significantly associated with mannose and lysosomal functional regulatory pathways, suggesting that this gene may play a key role in the molecular mechanism of the intervention in Example 1.

[0126] To verify the accuracy of the transcriptome sequencing results, qPCR was used to verify whether the LManV gene level in the gut of 30-day-old Drosophila fed in the example and control groups was upregulated. The results are as follows: Figure 2 After intervention in both the example and the comparative examples, the transcription level of LManV increased, and the upregulation effect of the example on LManV gene expression was greater than that in the comparative example.

[0127] 4. Autophagy gene transcription level analysis

[0128] Six biological replicates were used for each group, with midgut tissue from 30 Drosophila dissected in each replicate. RNAiso plus reagent was added. Phase separation was performed using chloroform, and RNA was precipitated using isopropanol. The precipitated RNA was washed with 75% ethanol and finally redissolved in 20 μL of DEPC water. The concentration of RNA was determined by NanoDrop assay and normalized to ensure the homogeneity of subsequent experiments. The normalized RNA samples were used to synthesize cDNA and quantified using a real-time quantitative PCR system. The relative expression level of the Act5c gene was calculated using the 2−ΔΔCT method, with the Act5c gene as an internal control. The primer information used is shown in Table 5, and the results of differentially expressed gene screening and analysis are shown in Table 6.

[0129] Table 5 Primer Sequences

[0130]

[0131] Table 6. Relative expression levels of autophagy gene mRNA

[0132]

[0133] qPCR was used to verify whether the samples promoted the transcriptional activation of autophagy-related genes. Table 6 shows that the transcriptional levels of Atg1, Atg14, Vps15, Atg9, Atg5, and Syx17 were decreased in the aged control group, while the expression of autophagy-related genes was upregulated in aged Drosophila after intervention in both the examples and the comparative examples. The upregulation effect of the examples on autophagy-related gene expression was greater than that in the comparative examples.

[0134] In summary, the Epimedium extract significantly increased the content of epimedium glycoside I, which could significantly upregulate the expression of lysosomal mannosidase (LManV), enhance autophagic flux, and promote the fusion of autophagosomes and lysosomes, thereby effectively clearing damaged intracellular components. This effect was verified in the Drosophila gut and human Caco-2 cells.

[0135] (iv) Toxicological experiment of Epimedium extract in Example 1

[0136] 4.1 Laboratory Animals

[0137] One hundred clean-grade SD rats were used in this experiment, half male and half female, with an initial weight of 180-220 g (male) and 160-200 g (female). All animals were acclimatized for one week under standard experimental conditions, with the room temperature maintained at 22±2℃, the relative humidity at 50±10%, and a 12-hour alternating light and dark lighting.

[0138] 4.2 Experimental Design and Grouping

[0139] 4.2.1 Acute oral toxicity test

[0140] Forty healthy SD rats, half male and half female, were randomly divided into a blank control group (0.5% CMC-Na) and the maximum dose group (5000 mg / kg bw) as described in Example 1, with 10 rats in each group. After fasting for 12 hours but with free access to water, the animals were given the corresponding drug solution or solvent in a single gavage at a volume of 10 mL / kg body weight. Immediate responses (such as activity, fur, respiration, secretions, and behavior) were observed for 4 hours after administration, followed by observation twice daily for 14 days. Any signs of toxicity, their onset and recovery time, and mortality were recorded. On day 14, all surviving animals were weighed, anesthetized with carbon dioxide inhalation, and euthanized for a systematic gross anatomical examination, focusing on changes in the morphology, color, and texture of major organs such as the heart, liver, spleen, lungs, kidneys, and stomach. Specific results are shown in Table 7.

[0141] 4.2.2 28-day repeated-dose toxicity test

[0142] Sixty healthy SD rats, half male and half female, were randomly divided into four groups according to body weight: a blank control group (0.5% CMC-Na), a low-dose group (100 mg / kg bw), a medium-dose group (300 mg / kg bw), and a high-dose group (1000 mg / kg bw), with 15 rats in each group. Males and females were housed separately. The rats were administered the drug by gavage at a dose of 10 mL / kg once daily in the morning for 28 consecutive days. The control group received an equal volume of the drug. Body weight and food intake were recorded twice weekly, and detailed clinical observations were conducted daily. On day 29 of the administration period, 10 animals (half male and half female) from each group were randomly sacrificed.

[0143] Hematological and serum biochemical tests were performed, including white blood cell count (WBC), red blood cell count (RBC), hemoglobin concentration (HGB), platelet count (PLT), alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), blood urea nitrogen (BUN), creatinine (CRE), total protein (TP), and albumin (ALB). Immediately after blood collection, the animal was dissected, and the heart, liver, spleen, lungs, kidneys, adrenal glands, thymus, testes (or ovaries), and uterus were rapidly removed. The organ coefficient (organ weight / final body weight × 100%) was calculated, and any histopathological changes, including degeneration, necrosis, inflammation, and hyperplasia, were observed and recorded.

[0144] The remaining 5 animals (half male and half female) entered a 14-day recovery period for observation, during which no drugs were administered. They were sacrificed on day 42 to assess the reversibility of the toxicity. Specific results are shown in Table 8.

[0145] Table 7 Results of Acute Oral Toxicity Tests

[0146]

[0147] Table 8: Key Indicators Results of the 28-Day Repeated-Dose Toxicity Study

[0148]

[0149] As shown in Tables 7-8, the Epimedium extract of Example 1 is non-toxic and harmless to human organs.

[0150] (V) Human trial of Epimedium extract from Example 1

[0151] 5.1 Sample Preparation

[0152] Test substance: Example 1 was encapsulated in opaque capsules by a third-party organization, with each capsule containing 250 mg of extract.

[0153] Placebo: Consists of starch capsules that are identical in appearance, odor, and weight to the test substance capsule.

[0154] Packaging and blinding: All capsules are packaged in a uniform manner and identified only by random numbers. The random number table is sealed and stored by statisticians who are not involved in the study execution and data analysis, and remains unblinded until the data is locked.

[0155] 5.2 Subject Screening

[0156] Inclusion criteria: ① Age 30-60 years; ② Body mass index (BMI) 18.5-24.0 kg / m²; ③ No long-term use of drugs (such as rapamycin, metformin, etc.) or health supplements affecting autophagy or metabolism within the past 3 months; ④ Willing to sign an informed consent form. Exclusion criteria: ① History of serious diseases of major organs such as liver, kidney, heart, and lungs; ② Pregnant or lactating women; ③ Known allergy to epimedium or related plants; ④ Recent history of surgery or planned surgery; ⑤ Other circumstances deemed unsuitable for participation by the investigators. A total of 60 eligible subjects were ultimately included and randomly assigned to the high-dose group, low-dose group, and placebo group in a 1:1:1 ratio using a computer-generated random number table.

[0157] 5.3 Test Design

[0158] This trial was a randomized, double-blind, placebo-controlled parallel-group design. The intervention period was 28 days.

[0159] High-dose group: Two test substance capsules (total dose 500 mg extract / day) were taken orally daily, one capsule in the morning and one in the evening.

[0160] Low-dose group: One test substance capsule orally daily (total dose 250 mg extract / day).

[0161] Placebo group: Two placebo capsules were taken orally daily, in the same manner as the high-dose group.

[0162] All participants returned to the research center for follow-up and sample collection at baseline (day 0) and at the end of the intervention period (day 29). During the study, participants were asked to maintain their daily diet and exercise habits and record any discomfort symptoms that may occur.

[0163] 5.4 Detection of autophagy markers in peripheral blood mononuclear cells

[0164] At baseline and on day 29, 10 mL of peripheral venous blood was collected from subjects in heparin anticoagulant tubes under fasting conditions. Peripheral blood mononuclear cells (PBMCs) were isolated within 2 hours, washed with pre-cooled PBS, and total protein was extracted using lysis buffer containing protease inhibitors. Protein concentration was determined using the BCA method. Equal volumes of protein samples were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and Western blotting analysis was performed after transfer to a membrane to calculate the LC3B-II / LC3B-I ratio (reflecting the level of autophagy formation) and the relative expression level of p62 protein (p62 / β-Actin, whose degradation reflects autophagic flux activity).

[0165] 5.5 Security Testing

[0166] Each visit includes a routine physical examination of the subject, including:

[0167] Vital signs and physical examination: Measure and record resting blood pressure, heart rate, body temperature, and weight at each visit.

[0168] Complete blood count: Using a fully automated blood analyzer to detect white blood cell count (WBC), red blood cell count (RBC), hemoglobin (HGB), platelet count (PLT), etc.

[0169] Serum biochemistry: Key liver function indicators [alanine aminotransferase (ALT), aspartate aminotransferase (AST)] and key kidney function indicators [blood urea nitrogen (BUN), creatinine (CRE)] were measured using a fully automated biochemical analyzer. Specific examination data are shown in Table 9.

[0170] Table 9. Physical Examination Results of Subjects

[0171]

[0172] As shown in Table 9, the Epimedium extract of Example 1 has good autophagy flux activity, can enhance cell autophagy function, and is safe and harmless to the human body.

[0173] (vi) Topical application experiment of Epimedium extract in Example 1

[0174] 6.1 Test substance and reference substance

[0175] The efficacy-enhancing cream contains Epimedium extract prepared by a patented method as its active ingredient, at a concentration of 3% (w / w). The cream base is an oil-in-water emulsion and does not contain other known potent anti-aging ingredients (such as retinol, high concentrations of vitamin C, etc.). The cream comprises the following components: 2.0 g Epimedium extract, 5.0 g glycerin, 3.0 g emulsifier (cetearearyl alcohol polyether-20), and deionized water to 100 g, homogenized and emulsified to obtain the Epimedium extract cream.

[0176] Placebo cream: Contains only the exact same cream base as the efficacy group, without epimedium extract. Both are identical in color, texture, and odor.

[0177] Packaging and blinding: All products were packaged in opaque aluminum tubes of the same size and identified only by a random three-digit code. The list of randomized codes was sealed and kept by statisticians independent of the study implementers until the data analysis was completed and the blinding was performed.

[0178] 6.2 Subject Screening and Grouping

[0179] Healthy female volunteers aged 35-55 with mild to moderate photoaging characteristics (manifested as fine lines and mild loss of skin elasticity) were selected through open recruitment. Exclusion criteria included: pregnant or breastfeeding women; those with severe skin diseases or active facial inflammation; those who had undergone chemical peels, laser treatments, or other facial cosmetic procedures within the past 3 months; those with known allergies to any ingredient in the investigational product; and those currently using medications or functional cosmetics that may affect skin condition. Sixty eligible participants were ultimately enrolled and randomly assigned in a 1:1 ratio to the efficacy group (using a cream containing 3% Epimedium extract) and the placebo group (using a base cream), with 30 participants in each group.

[0180] 6.3 Research Design and Usage Process

[0181] This study employed a randomized, double-blind, placebo-controlled, parallel-group design with a total duration of 8 weeks, including a baseline visit (day 0) and an endpoint visit (week 8). All participants were required to cleanse their faces 24 hours prior to each visit and avoid using any skincare products on the day of the visit. After completing all tests at the baseline visit, researchers distributed a uniformly numbered product and instructed participants to apply a sufficient amount (approximately 0.5 g) of the designated cream evenly to the entire face (with a focus on the eye and cheek areas) after cleansing morning and evening. Participants were required to use the same gentle cleansing product throughout the study and avoid using any other anti-aging or functional skincare products, while also keeping a product usage log and recording any discomfort.

[0182] 6.4 Evaluation Indicators and Testing Methods

[0183] 6.4.1 Key efficacy indicators (biomarkers related to autophagy and anti-aging)

[0184] Skin surface proteins were collected using a non-invasive keratinocyte peeling technique. At each visit, standard transparent tape was applied and peeled off with constant pressure on the lateral corner of the eye on the same side of the subject, repeated six times. The collected tape samples were stored at -80°C. The tape eluent was quantitatively analyzed using enzyme-linked immunosorbent assay (ELISA).

[0185] Autophagic flux markers: Quantitative determination of p62 protein and LC3B-II. Calculation of the LC3B-II / p62 ratio; an increase in this ratio is considered a comprehensive indicator of autophagic flux activation.

[0186] 6.4.2 Secondary efficacy indicators (measured by non-invasive instruments in skin physiology)

[0187] In a temperature- and humidity-controlled laboratory environment, after the subjects sat quietly for 20 minutes, the following professional skin testing instruments were used to measure the fixed marked points:

[0188] Skin wrinkles and texture: A fast optical imaging system is used to capture three-dimensional images of the crow's feet area around the eyes. The built-in software analyzes the images and outputs the wrinkle volume (mm³) and skin roughness Rz value (μm).

[0189] Skin elasticity and firmness: Using a skin elasticity tester, based on the principles of suction and stretching, the maximum displacement and rebound speed of the skin after being stretched are measured, and the skin elasticity R2 value (unitless) and bio-firmness R7 value (unitless) are calculated.

[0190] Skin barrier function and moisturizing: Transepidermal water loss (g / h·m²) was measured using a transepidermal water loss meter; stratum corneum water content (any conductivity unit) was measured using a skin moisture meter. Changes in skin biomarkers and physiological indicators are shown in Table 10.

[0191] Table 10 Changes in skin biomarkers and physiological indicators (x ± s)

[0192]

[0193] As shown in Table 10, the Epimedium extract of Example 1 enhances the autophagy function of cells and has good anti-wrinkle, firming and moisturizing effects after application, and can be used as a raw material for anti-aging products.

[0194] In summary, the Epimedium extract of the present invention, based on its effect of activating autophagy, can be used to prepare pharmaceuticals and skin care products.

[0195] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for preparing an epimedium extract that activates autophagy, characterized in that, Includes the following steps: S1. Wash fresh Epimedium, dry it, grind it into powder, and sift it to obtain Epimedium powder; S2. Add 10-50 times the weight of water to the Epimedium powder, stir well, and sterilize at high temperature to obtain the fermentation substrate; S3. Inoculate the fermentation substrate with a compound bacterial solution for fermentation. After the reaction is complete, sterilize to obtain Epimedium fermentation broth. The compound bacterial solution is a mixed bacterial solution of Lactobacillus plantarum, Lactobacillus acidophilus, and Lactobacillus casei. The Lactobacillus plantarum was purchased from Guangdong Provincial Microbial Culture Collection Center, with accession number GDMCC NO.64374. The Lactobacillus acidophilus was purchased from China General Microbial Culture Collection Center, with accession number CGMCC 1.1854. The Lactobacillus casei was purchased from Ningbo Taisto Biotechnology Co., Ltd., with catalog number TS277920. S4. Add a compound enzyme to the Epimedium fermentation broth for enzymatic hydrolysis. After the enzymatic hydrolysis is completed, inactivate the enzyme and spray dry to obtain the Epimedium extract that activates cell autophagy. The compound enzyme is pectin lyase and pullulanase.

2. The method for preparing the Epimedium extract for activating autophagy as described in claim 1, characterized in that, The viable count of the compound bacterial solution is 1.0 × 10⁻⁶. 9 –5.0×10 9 CFU / mL; the inoculation concentration of the compound bacterial solution is 0.7%-3% v / v, and the viable count ratio of Lactobacillus plantarum, Lactobacillus acidophilus and Lactobacillus casei bacterial solutions is (4-5):(0.2-1):(0.2-1).

3. The method for preparing the Epimedium extract for activating autophagy as described in claim 1, characterized in that, In step S3, the fermentation culture temperature is 25-35℃ and the time is 36-60h.

4. The method for preparing the Epimedium extract for activating autophagy as described in claim 1, characterized in that, The pectin lyase has an activity of 5000–20000 U / g and is added at a rate of 0.15–1.5% of the weight of the Epimedium fermentation broth; the pullulanase has an activity of 1000–8000 U / g and is added at a rate of 0.05–0.5% of the weight of the Epimedium fermentation broth; the enzymatic hydrolysis temperature is 40–60℃ and the time is 1–3 h.

5. An extract of Epimedium that activates autophagy, characterized in that, It is prepared by the method for preparing the Epimedium extract for activating autophagy as described in any one of claims 1-4.

6. The use of the Epimedium extract for activating autophagy as described in claim 5 in the preparation of pharmaceuticals and skin care products with anti-aging effects.

Images