Artificial culture method of phellinus igniarius as well as preparation method and application of phellinus igniarius extract

Abstract

The invention provides an artificial culture method of phellinus igniarius and a preparation method and application of a phellinus igniarius extract, and belongs to the technical field of fungus extracts. The invention provides an artificial culture method of phellinus igniarius, which comprises the following steps: carrying out shake culture on the phellinus igniarius by using a liquid culture medium, and preparing a phellinus igniarius extract after the phellinus igniarius is cultured for 14 days. According to the invention, artificially cultured phellinus igniarius is adopted as a raw material for preparation, compared with wild phellinus igniarius, the production cost is remarkably reduced, the phellinus igniarius extract obtained after extraction contains a plurality of active components, and compared with the previous research on a single component, the mixed components can play a better liver protection characteristic, and the liver protection effect is better. The protection effect on hepatocytes is realized through the anti-oxidation performance. Meanwhile, the phellinus igniarius extract prepared by the invention can also be used as a regulating protective agent for oxidative stress, inflammatory response, cell apoptosis and mitochondrial dysfunction, exerts the protection characteristic of liver cells, and lays a foundation for development of phellinus igniarius related liver protection products.

Description

Technical Field

[0001] The invention belongs to the technical field of fungus extracts, and specifically relates to an artificial cultivation method of Phellinus linteus and a preparation method and use of a Phellinus linteus extract. Background Art

[0002] Liver disease is a common and extremely harmful disease. A large number of clinical trials and animal studies have shown that persistent oxidative stress in the liver is crucial to the development of liver disease and is considered to be a key factor in the pathogenesis of acute and chronic liver disease. Alcohol, drugs and other factors are the causes of oxidative stress and ROS production. These factors can cause the accumulation of oxidative products responsible for ROS production, reduce the activity of antioxidant enzymes such as CAT, SOD, GSH-px and GST, and may also increase the gene transcription of ERRγ and CYP2E1, increase the level of lipid peroxidation, lead to liver oxidative stress, and may cause liver disease. Under oxidative stress conditions, several ARE genes and enzymes are activated, inhibiting oxidative stress responses and controlling liver diseases.

[0003] As a traditional Chinese medicine with a long history of more than 2,000 years, Phellinus igniarius has attracted extensive attention from scholars at home and abroad due to its unique pharmacological effects and high safety. Many studies have shown that Phellinus igniarius has multiple activities such as anti-tumor, anti-inflammatory, anti-injury and antioxidant. A large number of studies have shown that natural plant polysaccharides can be used as liver protectors by enhancing liver metabolism, reducing lipid peroxidation levels and inflammatory mediator expression, inhibiting oxidative stress and histopathological fibrosis, inducing hepatic stellate cell apoptosis and cycle arrest, and increasing antioxidant enzyme activity. Liu et al. confirmed that the extracellular polysaccharides in Phellinus igniarius fermentation broth play a protective role against acute liver injury in mice by prolonging the duration of the correction reflex, shortening the duration of recovery, and reducing the levels of ALT, AST and MDA. Chen et al. evaluated the protective effect of isolated Phellinus igniarius polysaccharides on acetaminophen-stimulated liver injury in mice by reducing the expression of CYP2E1 and the release of cytokines in the liver, increasing the level of phase II enzymes, showing liver repair properties and accelerating the metabolism of acetaminophen. It was found that its liver-protective activity was attributed to its antioxidant function, but there is little research on the overall effect of Phellinus igniarius extract. Summary of the invention

[0004] The present invention provides an artificial cultivation method of Phellinus linteus and a preparation method and use of a Phellinus linteus extract, which reduce the cultivation cost of Phellinus linteus. The Phellinus linteus extract has higher antioxidant activity and can exert better liver protection properties.

[0005] The present invention provides an artificial cultivation method of Phellinus linteus, comprising the following steps: inoculating Phellinus linteus into a liquid culture medium for shaking culture, wherein the shaking culture temperature is 26-32° C. and the time is 7-14 days;

[0006] Each liter of the liquid culture medium comprises 8 to 12 g of corn stalk powder and 14 to 18 g of potato glucose culture medium powder.

[0007] Preferably, the rotation speed of the shaking culture is 120-160 r / min.

[0008] The present invention also provides a method for preparing the mulberry linterus extract, comprising culturing and drying the mulberry linterus powder using the above-mentioned artificial cultivation method, performing ultrasonic-assisted extraction in sequence using an ethanol aqueous solution with a volume percentage of 95% and water, combining the extracts and centrifuging to collect the supernatant, wherein the supernatant contains the mulberry linterus extract.

[0009] Preferably, the mass volume ratio of the mulberry linterus powder to the ethanol aqueous solution and water is 0.8-1.5 g:100 mL;

[0010] Ultrasonic-assisted extraction was performed three times for each extraction solvent.

[0011] Preferably, the ultrasonic assisted extraction lasts for 1 to 2 hours each time, the power is 180 to 220 W, the ultrasonic frequency is 40 to 60 Hz, and the ultrasonic 3s interval is 1 to 3s.

[0012] The present invention also provides a Phellinus igniarius extract obtained by the above preparation method.

[0013] The present invention also provides the Phellinus linteus extract dry powder obtained by vacuum freeze-drying the Phellinus linteus extract.

[0014] The present invention also provides the use of the above-mentioned Phellinus linteus extract or the above-mentioned Phellinus linteus extract dry powder in the preparation of food or medicine having a protective effect on liver cell damage.

[0015] Preferably, the protective effect against liver cell damage includes at least one of the following: (1) antioxidant activity; (2) regulatory protection against oxidative stress, inflammatory response, cell apoptosis and mitochondrial dysfunction.

[0016] The present invention also provides a food or medicine having a protective effect on liver cell damage, wherein the active ingredient comprises the above-mentioned Phellinus linteus extract or the above-mentioned Phellinus linteus extract dry powder.

[0017] Beneficial effects: The present invention provides an artificial cultivation method of Phellinus igniarius, wherein the Phellinus igniarius is cultured in a liquid culture medium by shaking, and the Phellinus igniarius extract can be prepared when the culture reaches 14 days. The present invention adopts artificially cultured Phellinus igniarius as raw material for preparation, which significantly reduces the production cost compared with wild Phellinus igniarius. The Phellinus igniarius extract obtained after extraction contains multiple active ingredients such as polysaccharides, flavonoids, polyphenols, triterpenes, etc., and is tested to contain a variety of active ingredients. Compared with previous studies on single components, the mixed components can exert better liver protection properties, and their protective effect on liver cells is achieved through antioxidant properties. At the same time, the Phellinus igniarius extract prepared by the present invention can also be used as a regulator and protector of oxidative stress, inflammatory response, cell apoptosis, and mitochondrial dysfunction, exerting the protective properties of liver cells, and laying the foundation for the development of Phellinus igniarius related liver protection products. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a result diagram of the effect of mulberry linterus extract on the survival rate of liver cells detected by CCK8 method. In the figure, A shows the effect of different concentrations of PIE treatment on cell survival rate for 24 hours, B shows the effect of H2O2 concentration on cell survival rate, and C shows the effect of different concentrations of PIE pretreatment on the survival rate of L02 cells induced by H2O2 damage;

[0019] Figure 2 The results of the effect of Phellinus igniarius extract on AST (A), ALT (B) and LDH (C) in liver cells;

[0020] Figure 3 The results of the effect of Phellinus igniarius extract on SOD (A), CAT (B), GSH-Px (C) and MDA (D) in liver cells;

[0021] Figure 4 The results of the effect of Phellinus igniarius extract on ROS levels in cells; A in the figure is the ROS measurement result, and B is the ROS quantitative analysis result;

[0022] Figure 5 The results of the effect of Phellinus igniarius extract on the expression of Keap1 (A), Nrf2 (B), HO-1 (C), NQO1 (D) and GCLC (E) in cells;

[0023] Figure 6 The results of the effect of Phellinus igniarius extract on the expression of GST (A), GSR (B), Catalase (C), SOD1 (D), SOD2 (E), Gpx1 (F) and Gpx4 (G) in cells;

[0024] Figure 7 The results of the effect of Phellinus igniarius extract on the relative expression of inflammatory cytokines TNF-α (A), IL-6 (B) and IL-1β (C);

[0025] Figure 8 The results of the effect of Phellinus igniarius extract on cell apoptosis, A in the figure is the DAPI staining result, and B is the quantitative analysis result;

[0026] Fig. 9 This is the result diagram of the effect of Phellinus igniarius extract on apoptosis of L02 cells;

[0027] Fig.10 This is the result diagram of the effect of Phellinus igniarius extract on the gene of mitochondrial membrane potential;

[0028] Fig.11 This is a graph showing the effect of mulberry ignia extract on the relative expression levels of mitochondrial apoptosis pathway-related genes Caspase-3 / 8 / 9, BCL-2, and BAX. DETAILED DESCRIPTION

[0029] The present invention provides an artificial cultivation method of Phellinus linteus, comprising the following steps: inoculating Phellinus linteus into a liquid culture medium for shaking culture, wherein the shaking culture temperature is 26-32° C. and the time is 7-14 days;

[0030] Each liter of the liquid culture medium comprises 8 to 12 g of corn stalk powder and 14 to 18 g of potato glucose culture medium powder.

[0031] In a specific embodiment of the present invention, Phellinus igniarius is inoculated into the liquid culture medium for shaking culture. The strain is provided by Changbai Linfeng Fungi Co., Ltd. and preserved by the Key Laboratory of Molecular Enzymology Engineering of the Ministry of Education of Jilin University. The liquid culture medium of the present invention contains corn stalk powder, wherein the particle size of the corn stalk powder can pass through a 100-mesh sieve; the potato glucose culture medium powder is purchased from Qingdao High-tech Industrial Park Haibo Biotechnology Co., Ltd., with the item number HB0233-4; in the preparation of the liquid culture medium in the embodiment, any mass of 8-12g corn stalk powder and 14-18g potato glucose culture medium powder can be selected for preparation, wherein the corn stalk powder can be 10.4g, and the potato glucose culture medium powder can be 15.6g.

[0032] In a specific embodiment of the present invention, the shaking culture is carried out in a triangular flask, such as the liquid volume in the triangular flask can be 49%, the pH value is 6.5, and then the culture is carried out at a rotation speed of 120 to 160 r / min, and the rotation speed can be 148.9 r / min; the culture time can be 7 to 14 days, such as 14 days; wherein the shaking culture temperature can be any temperature between 26 and 32°C, such as 29.2°C in the embodiment,

[0033] The present invention also provides a method for preparing the mulberry linterus extract, comprising culturing and drying the mulberry linterus powder using the above-mentioned artificial cultivation method, performing ultrasonic-assisted extraction in sequence using an ethanol aqueous solution with a volume percentage of 95% and water, combining the extracts and centrifuging to collect the supernatant, wherein the supernatant contains the mulberry linterus extract.

[0034] The present invention uses mulberry powder as a raw material for ultrasonic-assisted extraction, wherein the mass volume ratio of mulberry powder to 95% ethanol aqueous solution can be 0.8-1.5g:100mL, such as 1g:100mL; and the 95% ethanol aqueous solution is used for ultrasonic-assisted extraction 3 times. In a specific embodiment of the present invention, when using 95% ethanol aqueous solution for ultrasonic-assisted extraction, the time of each extraction is 2h, the power is 180-220W, the ultrasonic frequency is 50Hz, and the ultrasonic 3s interval is 1-3s; and the first extraction and the second extraction are collected separately, and then water is directly added after the third extraction for 3 extractions, and the mass volume ratio of mulberry powder to water is 1g:100mL. In a specific embodiment of the present invention, when using water for ultrasonic-assisted extraction, the time of each extraction is 2h, the power is 200W, the ultrasonic frequency is 50Hz, and the ultrasonic 3s interval is 3s.

[0035] The present invention combines the extracts from each ultrasonic-assisted extraction, and then collects the supernatant by centrifugation, wherein the centrifugation needs to be performed under low temperature conditions, such as centrifugation at 4°C, the centrifugation speed can be 10000r / min, and the time can be 10min.

[0036] The present invention also provides a Phellinus igniarius extract obtained by the above preparation method.

[0037] The Phellinus igniarius extract of the present invention has been tested to contain multiple active ingredients such as polysaccharides, flavonoids, polyphenols, triterpenes, etc. Compared with previous studies on single components, the mixed components can exert better liver-protecting properties.

[0038] The present invention also provides the Phellinus linteus extract dry powder obtained by vacuum freeze-drying the Phellinus linteus extract.

[0039] In a specific embodiment of the present invention, the supernatant containing the mulberry linterus extract is subjected to rotary evaporation and then the extract is freeze-dried using a vacuum freeze dryer, and the rotary evaporation temperature can be 60-80°C and the rotation speed can be 80-120r / min, thereby obtaining the mulberry linterus extract dry powder.

[0040] The present invention also provides the use of the above-mentioned Phellinus linteus extract or the above-mentioned Phellinus linteus extract dry powder in the preparation of food or medicine having a protective effect on liver cell damage.

[0041] The examples of the present invention demonstrate that the protective effect against liver cell injury includes at least one of the following: (1) antioxidant activity; and (2) regulatory protection against oxidative stress, inflammatory response, cell apoptosis, and mitochondrial dysfunction.

[0042] The present invention also provides a food or medicine having a protective effect on liver cell damage, wherein the active ingredient comprises the above-mentioned Phellinus linteus extract or the above-mentioned Phellinus linteus extract dry powder.

[0043] In order to further illustrate the present invention, an artificial cultivation method of Phellinus linteus and a preparation method and use of Phellinus linteus extract provided by the present invention are described in detail below in conjunction with embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0044] Example 1

[0045] Artificial cultivation method of Phellinus igniarius: Weigh 1.04g corn stalk powder and 1.56g potato glucose medium, place in 100mL water, sterilize and inoculate, and culture for 14 days at 29.2℃, 49% liquid volume, pH=6.5, and 148.9r / min to fully accumulate its effective ingredients. After cultivation, separate the supernatant and Phellinus igniarius mycelium by high-speed centrifugation, dry the mycelium in a 40℃ oven, grind it after drying, sieve it through 100 mesh, and store its fine powder at -20℃.

[0046] Method for obtaining Phellinus igniarius extract: weigh 20 g of artificially cultured Phellinus igniarius fine powder sample, add 200 mL of 95% ethanol solution, mix well, extract at 60°C, 200 W for 2 h, repeat three times, then add 200 mL of water, extract at 60°C, 200 W for 2 h, repeat three times, combine the extracts, collect the supernatant by centrifugation, rotary evaporation and freeze-dry the extract in a vacuum freeze dryer to obtain Phellinus igniarius extract powder.

[0047] Experiments were conducted on the Phellinus linteus extract powder in Example 1:

[0048] 1. Detection of hepatocyte survival rate under the action of Phellinus igniarius extract using CCK8 method

[0049] (1) Collect L02 cells and add 200 μL per well to a 96-well plate (1×10 4 Each well was added with 200 μL PBS solution and placed in an incubator until the cells were completely attached to the wall. The culture medium was discarded, and 6 replicate wells were set up in each group. Complete culture medium [fetal bovine serum and DMEM culture medium (with 1% penicillin-streptomycin)] containing different concentrations of phellinus linteus extract (0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5 mg / mL) was added, and cultured for 24 hours. The cell survival rate was detected by CCK-8 method.

[0050] (2) After the cells were plated as described above, the old culture medium was discarded and H2O2 solutions of different concentrations (0, 100, 200, 300, 400, 500, 600, 700, 800, 900 μmol / L) were added to culture the cells for 12 h. The culture medium was then replaced and the cell survival rate was detected using the CCK-8 method.

[0051] (3) After the above-mentioned plating method, the old culture medium was discarded, and complete culture medium containing different concentrations of Phellinus linteus extract was added first, and the cells were cultured for 24 hours. Then, different concentrations of H2O2 solution were added to culture the cells for 12 hours, and the cell survival rate was detected by CCK-8 method.

[0052]

[0053] As the concentration of Phellinus igniarius extract increased, the survival rate of L02 cells showed no significant change trend, indicating that there was no damage to the cells at the experimental concentration ( Figure 1 (A). H2O2 can significantly reduce the survival rate of L02 cells in a dose-dependent manner. When 600 μmol / L H2O2 was added, the cell survival rate was (53.83±2.37)%, reaching the median lethal dose ( Figure 1 After the cells were treated with H2O2 alone for 12 hours, the cell survival rate was (52.32±2.50)%, and when the cells were pretreated with Phellinus igniarius extract for 24 hours, the cell survival rate was improved to a certain extent. When the drug concentration was 0.1, 0.2, 0.3, 0.4, and 0.5 mg / mL, the cell survival rates were (60.64±2.18)%, (69.76±2.75)%, (74.67±1.54)%, (84.38±2.86)%, (84.09±2.31)%, respectively. Figure 1 Middle C), indicating that pretreatment with Phellinus igniarius extract can effectively inhibit the damaging effect of H2O2 on cells and improve cell survival rate.

[0054] 2. Use AST, ALT and LDH kits to detect the protective effect of Phellinus igniarius extract on hepatocytes

[0055] The cells were collected and counted according to the subculture method and added to six-well plates (3×10 4 / mL), and the cells were cultured until they adhered. Complete medium was added to the control group and the H2O2 group, and complete medium containing different concentrations of Phellinus linteus extract (0.1, 0.2, 0.4 mg / mL) was added to the drug group, and the cells were cultured for 24 hours. The culture medium was discarded, complete medium was added to the control group, and 600 μmol / L H2O2 solution was added to the H2O2 group and the drug group to culture the cells for 12 hours. After washing and digesting the cells, the ALT, AST, and LDH kit instructions were followed.

[0056] The results are as follows Figure 2 As shown in the figure, after adding H2O2, the ALT and AST contents in the cell lysis supernatant increased by 127.82% and 169.75%, respectively, and the LDH leakage increased by 37.02%. After pretreatment with Phellinus igniarius extract, the contents of the three decreased in a dose-dependent manner. When the concentration of Phellinus igniarius extract was 0.4 mg / mL, the ALT, AST and LDH contents decreased by 30.75%, 46.48% and 17.43% respectively compared with the H2O2 group after 24 hours of pretreatment. The results showed that Phellinus igniarius extract inhibited the release of AST, ALT and LDH, and had a protective effect on L02 cell damage.

[0057] 3. Use SOD, CAT, GSH-Px and MDA kits to detect the protective effect of Phellinus igniarius extract on hepatocytes

[0058] The method was the same as that of Experiment 2. After washing and digesting the cells, the procedures were performed according to the instructions of the SOD, CAT, GSH-Px and MDA kits.

[0059] The results are as follows Figure 3 As shown in the figure, after adding H2O2, the contents of SOD, CAT, and GSH-Px decreased by 51.17%, 47.44%, and 62.87%, respectively, and the content of MDA increased by 126.79%. After pretreatment with Phellinus igniarius extract, the contents of antioxidant enzymes SOD, CAT, and GSH-Px increased in a dose-dependent manner, and the content of malondialdehyde (MDA) decreased in a dose-dependent manner. When the concentration of Phellinus igniarius extract was 0.4 mg / mL, compared with the H2O2 group after pretreatment for 24 hours, the contents of SOD, CAT, and GSH-Px increased by 75.05%, 57.76%, and 97.44%, respectively, and the content of MDA decreased by 23.62%. This shows that after pretreatment of cells with Phellinus igniarius extract for 24 hours, it can significantly change the cell oxidative damage caused by H2O2 stimulation, which has certain research significance.

[0060] 4. ROS level detection

[0061] The cell culture method was the same as in Experiment 2. The culture medium was discarded, the cells were washed 3 times with PBS, and 1 mL of diluted fluorescent probe DCFH-DA (Shanghai Biotech Co., Ltd.) was added to each well. After being placed at 37°C for 20 min, the cells were washed twice with DMEM culture medium, observed under a fluorescence microscope, and analyzed with Image J.

[0062] The results are as follows Figure 4As shown, the fluorescence intensity in the control group cells was weak, indicating that the intracellular ROS level was low. The green fluorescence of the H2O2 treatment group was significantly enhanced, indicating that H2O2 caused oxidative damage to L02 cells. Compared with the H2O2 treatment group, the fluorescence intensity of the group pretreated with Phellinus igniarius extract decreased by 39.50%, 45.72% and 65.49%, respectively, in a dose-dependent manner. This shows that after pretreatment with Phellinus igniarius extract, ROS generation can be effectively inhibited, cell oxidative damage caused by ROS can be reduced, and liver protection properties can be exerted.

[0063] 5. Antioxidant gene expression detection

[0064] The cell culture method was the same as that in Experiment 2. qPCR was used to detect the relative expression of genes. The sequences are shown in Table 1.

[0065] Table 1 Primer sequences

[0066]

[0067]

[0068] The results are as follows Figure 5 , 6 As shown in the figure, after adding H2O2, the relative expression levels of genes Keap1, Nrf2, HO-1, NQO1, GCLC, GST, GSR, Catalase, SOD2, Gpx1 and Gpx4 related to oxidative stress were significantly reduced. After adding phellus igniarius extract for pre-protection, compared with the H2O2 group, except for the relative expression level of SOD1, the other genes were significantly increased, proving that phellus igniarius extract can effectively reduce L02 cell damage caused by oxidative stress.

[0069] 6. Effect of Phellinus igniarius extract on the relative expression of inflammatory cytokines

[0070] The cell culture method was the same as that in Experiment 2, and the relative gene expression was detected by qPCR using the primers in Table 1.

[0071] The results are as follows Figure 7As shown in the results, after pretreatment with Phellinus igniarius extract, the expression of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL-1β) caused by oxidative stress can be significantly reduced. Compared with the control group, after adding H2O2, the relative expression of TNF-α, IL-6 and IL-1β increased by 61%, 116% and 120.79%, respectively. After pretreatment with Phellinus igniarius extract, the relative expression of inflammatory cytokines decreased. When the concentration of Phellinus igniarius extract was 0.4 mg / mL, the relative expression of TNF-α, IL-6 and IL-1β decreased by 23.60%, 25.46% and 36.32%, respectively, compared with the H2O2 group after 24 hours of pretreatment. Therefore, it can be inferred that Phellinus igniarius extract has a hepatoprotective effect, and its effect may be achieved by downregulating the expression of inflammatory cytokines and inhibiting hepatocyte necrosis.

[0072] 7. Effect of Phellinus igniarius extract on cell apoptosis

[0073] The cell culture method was the same as that in Experiment 2, and cell apoptosis was detected using DAPI kit and flow cytometry.

[0074] like Figure 8 As shown in A, the cells in the control group had normal morphology and weak blue fluorescence. The proportion of live cells in the H2O2 group decreased, and granular apoptotic bodies appeared in the cells. After treatment with the extract of Phellinus igniarius, the fluorescence intensity was significantly weakened, and the number of apoptotic cells decreased in a dose-dependent manner. Figure 8 As shown in Figure B, the fluorescence intensity of the H2O2 group was 4.71 times higher than that of the control group, while after pretreatment with Phellinus igniarius extract, the fluorescence intensity decreased by 8.44%, 47.20% and 59.60%, respectively. Fig. 9 As shown in the results, the apoptosis rate of cells reached 25.9% after adding H2O2, which was significantly higher than that of the control group. However, after adding different concentrations of Phellinus igniarius extract, the apoptosis rate of cells showed a downward trend. In summary, Phellinus igniarius extract can reduce the apoptosis of L02 cells and exert the protective properties of liver cells.

[0075] 8. Effects of Phellinus igniarius extract on mitochondrial membrane potential and expression of genes related to mitochondrial apoptosis pathway

[0076] The cell culture method was the same as that in Experiment 2, and the mitochondrial membrane potential detection kit and qPCR method were used to detect gene expression. Fig.10As shown, the cells in the control group were not treated with drugs. When the JC-1 probe entered the cells, red fluorescence was observed, indicating that the cell structure was intact and the mitochondrial membrane potential was normal. The H2O2-treated group showed an increase in green fluorescence and a decrease in red fluorescence, indicating that H2O2 induced oxidative damage to L02 cells, decreased mitochondrial membrane potential, and led to cell apoptosis. When different concentrations of Phellinus igniarius extract were added, compared with the H2O2 group, the red fluorescence in the cells gradually increased, the green fluorescence gradually decreased, and the mitochondrial membrane potential increased, indicating that the addition of Phellinus igniarius extract can effectively alleviate the phenomenon of decreased mitochondrial membrane potential caused by cellular oxidative damage. Fig.11 As shown in the figure, after H2O2 was added, the expression of Caspase-3 / 8 / 9 and BAX in the cells increased significantly, while the expression of BCL-2 decreased, indicating that H2O2 induced L02 cell apoptosis through the mitochondrial apoptosis pathway. After adding different concentrations of Phellinus igniarius extract, compared with the H2O2 group, the expression of Caspase-3 / 8 / 9 and BAX decreased significantly, while the expression of BCL-2 increased, indicating that Phellinus igniarius extract may inhibit hepatocyte apoptosis by regulating the mitochondrial apoptosis pathway, thereby exerting liver protection properties.

[0077] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. An artificial cultivation method of Phellinus linteus, characterized in that: The method comprises the following steps: inoculating Phellinus linteus into a liquid culture medium for shaking culture, wherein the shaking culture temperature is 26 to 32° C. and the time is 7 to 14 days; Each liter of the liquid culture medium comprises 8 to 12 g of corn stalk powder and 14 to 18 g of potato glucose culture medium powder.

2. The artificial culture method according to claim 1, characterized in that: The rotation speed of the shaking culture is 120-160 r / min.

3. A method for preparing a Phellinus linteus extract, characterized in that: The invention comprises mulberry linterus powder cultivated and dried by the artificial cultivation method of claim 1 or 2, ultrasonic-assisted extraction using 95% ethanol aqueous solution and water in sequence, combining the extracts and collecting the supernatant by centrifugation, wherein the supernatant contains mulberry linterus extract.

4. The preparation method according to claim 3, characterized in that: The mass volume ratio of the mulberry linterus powder to the ethanol aqueous solution and water is 0.8-1.5 g:100 mL; Ultrasonic-assisted extraction was performed three times for each extraction solvent.

5. The preparation method according to claim 3 or 4, characterized in that: The ultrasonic assisted extraction lasts for 1 to 2 hours each time, the power is 180 to 220 W, the ultrasonic frequency is 40 to 60 Hz, and the ultrasonic 3s interval is 1 to 3s.

6. The Phellinus linteus extract obtained by the preparation method according to any one of claims 3 to 5.

7. The Phellinus linteus extract dry powder obtained by vacuum freeze-drying the Phellinus linteus extract according to claim 6.

8. Use of the Phellinus linteus extract according to claim 6 or the Phellinus linteus extract dry powder according to claim 7 in the preparation of food or medicine having a protective effect on liver cell damage.

9. The use according to claim 8, characterized in that: The protective effect against liver cell injury includes at least one of the following: (1) antioxidant activity; (2) regulatory protection against oxidative stress, inflammatory response, cell apoptosis and mitochondrial dysfunction.

10. A food or medicine having a protective effect on liver cell damage, characterized in that: The active ingredient includes the Phellinus linteus extract according to claim 6 or the Phellinus linteus extract dry powder according to claim 7.

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