Separation process and application of novel dipeptide Flolutsin C with anti-liver-injury activity in Helichrysum luteo-virens

Abstract

The invention discloses a separation process and application of novel dipeptide Flolutsin C with anti-liver injury activity in Chaetotrichum luteo-virens. The specific method comprises the following seven steps: extraction, QuikSep PS medium-pressure chromatography coarse separation, anti-liver injury active component preliminary screening, Megress C18 reversed-phase preparation column preparation, anti-liver injury active component re-screening, ACE Excel C18AR reversed-phase preparation column preparation and anti-liver injury in-vitro activity evaluation. A solvent and a separation material used in the process can be recycled, so that relatively low separation and preparation cost is ensured, and high-pressure preparative chromatographic separation can ensure that the purity of the product is greater than 95%; the activity screening method is simple, and the quality is stable and controllable; the experimental operation is simple, the cost is low, the quality is controllable, and the result is visual and easy to judge; the obtained anti-liver-injury active compound can be used for preparing anti-non-alcoholic fatty liver disease drugs. The preparation process provided by the invention can realize large-scale production.

Description

Technical Field

[0001] The invention relates to the technical field of separation of a substance with anti-liver damage activity in Pleurotus eryngii, and in particular to a separation process and application of a new dipeptide Flolutsin C with anti-liver damage activity in Pleurotus eryngii. Background Art

[0002] With the change of modern lifestyle and the improvement of living standards, the metabolic burden of the human liver is gradually increasing. Liver damage caused by excessive intake of high-fat and high-sugar foods is an important cause of non-alcoholic fatty liver disease. Currently, 30% of adults in the world suffer from non-alcoholic fatty liver disease, and the trend is obvious among younger people. The prevalence of non-alcoholic fatty liver disease in my country has also reached 15%, and has been on an upward trend in the past decade, but there are no approved clinical drugs so far. Therefore, discovering lead compounds with anti-liver injury activity from multiple pathways has become a hot topic in current research.

[0003] Floccularia luteovirens, belonging to the genus Floccularia of the family Agaricus (Agaricaceae), is commonly known as yellow mushroom and was formerly known as yellow-green honey fungus. It is a representative of rare wild mushrooms in alpine grasslands. As an edible fungus, it has the effects of immunomodulation, digestion promotion, antioxidant, blood pressure reduction, anti-inflammatory, and anti-liver damage. However, there are few studies on the chemical components of Floccularia luteovirens, and the material basis of its efficacy is still unclear. In order to further accelerate the quality evaluation, production and sales of Floccularia luteovirens and the research and development of related new drugs, it is necessary to explore its potential anti-liver damage active ingredients. Summary of the invention

[0004] Based on the above technical problems, the purpose of the present invention is to provide a separation process and application of a new dipeptide Flolutsin C with anti-liver damage activity in Pleurotus eryngii.

[0005] The present invention provides a separation process for protecting the new dipeptide Flolutsin C with anti-liver damage activity in Pleurotus eryngii, which specifically comprises the following steps:

[0006] Step 1, extraction: after the fresh sample of the yellow-green curly mushroom is roughly crushed, it is extracted with 95% ethanol at room temperature according to a solid-liquid ratio of 1g:5mL-100mL, and the extraction is performed 2-4 times, each time for 12-24 hours, and the filtrate is filtered and combined, that is, filtrate A, and the filtrate A is dried under reduced pressure to a paste to obtain the yellow-green curly mushroom ethanol extract extract; the yellow-green curly mushroom ethanol extract extract is extracted with petroleum ether and water in a volume ratio of 1:1, and the aqueous phase is concentrated. The sample is mixed according to the amount of silica gel: the amount of the aqueous phase = 3:1 and dried under reduced pressure to obtain the yellow-green curly mushroom water phase mixed sample;

[0007] Step 2, QuikSep PS medium-pressure chromatography crude fractionation: The sample of the yellow-green curcuma water phase mixture is separated by medium-pressure chromatography equipped with QuikSep PS, and detected by an ultraviolet detector with a detection wavelength of 210 nm. Six main fractions are collected according to the chromatographic peak shape, and then each fraction is dried under reduced pressure to obtain six component samples of Fr1, Fr2, Fr3, Fr4, Fr5, and Fr6;

[0008] Step 3, initial screening of anti-liver injury active components: each of the above 6 component samples was prepared into a sample solution with a concentration of 5 mg / mL using DMSO; human liver cancer cells HepG2 were cultured at a concentration of 2.5×10 5 The cells were inoculated in a 6-well plate at a density of 100 μM, and after they adhered to the wall and grew to the logarithmic growth phase, each component and oleic acid with a working concentration of 600 μM were added to the 6-well plate and incubated for 24 hours; after 24 hours, the culture medium was discarded, the cells were fixed with 4% formaldehyde for 15 minutes, the residual 4% formaldehyde was washed off with distilled water, the cells were rinsed with isopropanol for 20 seconds, and Oil Red O staining was performed for 40 minutes; after 40 minutes, the residual Oil Red O staining solution was washed off with distilled water; the cells were soaked in isopropanol for 40 minutes to dissolve the Oil Red O solution, the multifunctional microplate reader was turned on and preheated for 5 minutes, the absorbance of each component was detected at a wavelength of 510 nm, and the anti-liver injury activity of each component was quantitatively evaluated, and finally it was determined that the component Fr5 had the best anti-liver injury activity;

[0009] Step 4, preparation of Megress C18 reverse phase preparative column: add 5 times the volume of methanol to dissolve component Fr5, then add 2 times the volume of silica gel to mix and dry under reduced pressure to obtain Fr5 mixed sample; separate the Fr5 mixed sample by Megress C18 preparative chromatography, number the recovered components and dry under reduced pressure to obtain 15 component samples including Fr5-1, Fr5-2, Fr5-3, Fr5-4, Fr5-5, Fr5-6, Fr5-7, Fr5-8, Fr5-9, Fr5-10, Fr5-11, Fr5-12, Fr5-13, Fr5-14 and Fr5-15;

[0010] Step 5, rescreening of anti-liver injury active components: each of the 15 component samples was prepared into a sample solution with a concentration of 5 mg / mL using DMSO; human liver cancer cells HepG2 were cultured at a concentration of 2.5×10 5The cells were inoculated in a 6-well plate at a density of 100 μM. After the cells adhered to the wall and grew to the logarithmic growth phase, each component and oleic acid at a working concentration of 600 μM were added to the 6-well plate and incubated for 24 hours. After 24 hours, the culture medium was discarded, the cells were fixed with 4% formaldehyde for 15 minutes, the residual 4% formaldehyde was washed off with distilled water, and the cells were rinsed with isopropanol for 20 seconds. Oil Red O staining was performed for 40 minutes. After 40 minutes, the residual Oil Red O stain was washed off with distilled water. The cells were soaked in isopropanol for 40 minutes to dissolve the Oil Red O solution. The multifunctional microplate reader was turned on and preheated for 5 minutes. The absorbance of each component was detected at a wavelength of 510 nm, and the anti-liver injury activity of each component was quantitatively evaluated. Finally, it was determined that the component Fr5-9 had the best anti-liver injury activity.

[0011] Step 6, preparation of ACE Excel C18 AR reverse phase preparative column: add 5 times the volume of methanol and water to dissolve component Fr5-9, wherein the volume ratio of methanol to water is 2:3, then add 2 times the volume of silica gel to mix well and dry under reduced pressure to obtain Fr5-9 mixed sample; separate the Fr5-9 mixed sample on ACE Excel C18 AR preparative chromatography and dry under reduced pressure to obtain sample Fr5-9-3;

[0012] Step 7, in vitro evaluation of anti-liver injury activity: The sample Fr5-9-3 was prepared into a sample solution with a concentration of 1 mM using DMSO; human liver cancer cells HepG2 were cultured at a concentration of 2.5×10 5 The cells were inoculated in a 6-well plate at a density of 100 μg / cm2 and grown to the logarithmic growth phase after adherence to the wall. The Fr5-9-3 sample solution and oleic acid with a working concentration of 600 μM were added to the 6-well plate and incubated for 24 hours. After 24 hours, the culture medium was discarded, the cells were fixed with 4% formaldehyde for 15 minutes, the residual 4% formaldehyde was washed off with distilled water, and the cells were rinsed with isopropanol for 20 seconds. The cells were stained with Oil Red O for 40 minutes, and the residual Oil Red O dye was washed off with distilled water after 40 minutes. The cells were soaked in isopropanol for 40 minutes to dissolve the Oil Red O solution, the multifunctional microplate reader was turned on and preheated for 5 minutes, and the absorbance was detected at a wavelength of 510 nm. It was determined that Fr5-9-3 had in vitro anti-liver injury activity and was named Flolutsin C. The chemical structure of Flolutsin C is:

[0013]

[0014] Furthermore, in steps 1, 2, 4 and 6, the conditions for reduced pressure drying are: vacuum degree 50-250 mbar, temperature 40-60°C.

[0015] Furthermore, in step 2, the medium-pressure chromatographic separation operating parameters of the QuikSep PS column are: the chromatographic column size is 460×49 mm, the stationary phase is QuikSep PS, the mobile phase A is water, B is methanol, the chromatographic conditions are 0-150 minutes, 100% A, 150-500 minutes, 0-70% B; the flow rate is 40 mL / min, and the detection wavelength is 210 nm.

[0016] Furthermore, in step 3, the anti-liver injury active components were initially screened into: normal group, model group, and drug administration group. The normal group only added DMSO at the same concentration as the drug administration group, the model group only added oleic acid at a working concentration of 600 μM, and the drug administration group was 6 components + DMSO + 600 μM oleic acid; other conditions remained consistent.

[0017] Furthermore, in step 4, the high pressure chromatography separation working parameters of the Megress C18 column are: the column size is 250×20 mm; the mobile phase A is water, B is methanol, the chromatographic conditions are 0 to 60 minutes, 0 to 40% B; the flow rate is 19 mL / min, and the detection wavelength is 210 nm.

[0018] Furthermore, in step 5, the anti-liver injury active components were further screened into: normal group, model group, and drug administration group. The normal group only added DMSO at the same concentration as the drug administration group, the model group only added oleic acid at a working concentration of 600 μM, and the drug administration group was 15 components + DMSO + 600 μM oleic acid; other conditions remained consistent.

[0019] Furthermore, in step 6, the high pressure chromatography separation working parameters of the ACE Excel C18 AR column are: the size of the chromatographic column is 250×10 mm; the mobile phase A is water, B is acetonitrile, the chromatographic conditions are 0 to 30 minutes, 8% B; the flow rate is 19 mL / min, and the detection wavelength is 210 nm.

[0020] Furthermore, in step 7, the in vitro evaluation of anti-liver injury activity is divided into: a normal group, a model group, and a drug-treated group. The normal group only adds DMSO at the same concentration as the drug-treated group, the model group only adds oleic acid at a working concentration of 600 μM, and the drug-treated group is sample Fr5-9-3+DMSO+600 μM oleic acid; other conditions remain the same.

[0021] The present invention also protects the use of the new dipeptide Flolutsin C with anti-liver damage activity in the above-mentioned Pleurotus eryngii, wherein the new dipeptide Flolutsin C is used as an effective ingredient in combination with any acceptable carrier for preparing anti-liver damage medicines.

[0022] The novel dipeptide Flolutsin C is optimized for preparing drugs for treating non-alcoholic fatty liver disease.

[0023] Compared with the existing technology, the present invention has the following beneficial effects:

[0024] (1) The present invention has low cost and high product purity

[0025] The solvents used for extraction, QuikSep PS medium-pressure column and reverse-phase chromatography column separation can all be recycled; the materials used for medium-pressure separation and reverse-phase chromatography separation can all be reused. The recycled solvents and reused separation materials ensure a relatively low separation and preparation cost. High-pressure preparative chromatography separation can ensure that the product purity is greater than 95%.

[0026] (2) The activity screening method is simple and the quality is stable and controllable

[0027] After Oil Red O staining, the anti-liver injury activity of the sample was detected using an enzyme marker. The experimental operation is simple, the cost is low, the quality is controllable, the results are intuitive, and easy to judge. The obtained anti-liver injury active compounds can be used to develop and prepare therapeutic drugs for non-alcoholic fatty liver disease.

[0028] (3) The preparation process of the present invention can realize large-scale production

[0029] The raw material requirements are not high and the cost is low. Generally, wild or commercially available yellow-green curly mushrooms can be used, and it is easy to prepare in batches; 95% ethanol cold soaking extraction at room temperature is easy to operate; QuikSep PS medium-pressure column crude separation is used for separation, and the separation material can be installed in a medium-pressure column chromatography system, which is easy to achieve scale; the reversed-phase preparative liquid chromatography used in separation and purification is also very suitable for large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a medium pressure chromatographic separation diagram of the mixed sample of the yellow-green Pleurotus eryngii extract of the present invention;

[0031] Figure 2 This is a screening diagram for the anti-liver injury activity of the components Fr1 to Fr6 of Pleurotus eryngii of the present invention;

[0032] Figure 3 This is a Megress C18 reverse phase preparative liquid chromatography separation diagram of the target component Fr5 of Pleurotus eryngii of the present invention;

[0033] Figure 4 This is a screening diagram for the anti-liver injury activity of the components of Pleurotus eryngii Fr5-1 to Fr5-15 of the present invention;

[0034] Figure 5 This is the ACE Excel C18 AR reverse phase preparative liquid chromatography separation diagram of the target component Fr5-9 of Pleurotus eryngii of the present invention;

[0035] Figure 6This is a graph for evaluating the in vitro anti-liver injury activity of the novel dipeptide Flolutsin C of the present invention;

[0036] Figure 7 This is a high-resolution mass spectrum of the novel dipeptide Flolutsin C of the present invention;

[0037] Figure 8 The novel dipeptide Flolutsin C of the present invention 1 H-NMR nuclear magnetic spectrum;

[0038] Fig. 9 The novel dipeptide Flolutsin C of the present invention 13 C-NMR nuclear magnetic spectra;

[0039] Fig.10 This is the DEPT NMR image of the new dipeptide Flolutsin C of the present invention;

[0040] Fig.11 This is the HSQC two-dimensional NMR image of the new dipeptide Flolutsin C of the present invention;

[0041] Fig.12 This is the HMBC two-dimensional nuclear magnetic resonance image of the new dipeptide Flolutsin C of the present invention;

[0042] Fig.13 This is the COSY two-dimensional NMR image of the new dipeptide Flolutsin C of the present invention;

[0043] Fig.14 It is the NOESY two-dimensional nuclear magnetic resonance image of the new dipeptide Flolutsin C of the present invention;

[0044] Fig.15 This is the infrared spectrum of the new dipeptide Flolutsin C of the present invention;

[0045] Fig.16 This is the UV spectrum of the new dipeptide Flolutsin C of the present invention;

[0046] Fig.17 This is the optical rotation test diagram of the new dipeptide Flolutsin C of the present invention;

[0047] Fig.18 It is the chemical structural formula of the novel dipeptide Flolutsin C of the present invention. DETAILED DESCRIPTION

[0048] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0049] Example 1

[0050] The separation process of the new dipeptide Flolutsin C with anti-liver damage activity from Pleurotus eryngii specifically comprises the following steps:

[0051] Step 1, extraction: take 150g of fresh yellow-green curly hair mushroom sample, roughly chop it, extract it with 95% ethanol at room temperature according to the solid-liquid ratio of 1g:5mL, extract it twice, each time for 12 hours, filter and combine the filtrate, that is, filtrate A, and reduce pressure and dry the filtrate A to a paste to obtain the yellow-green curly hair mushroom ethanol extract extract; the yellow-green curly hair mushroom ethanol extract extract is extracted with petroleum ether and water in a volume ratio of 1:1, the aqueous phase is concentrated, the sample is mixed according to the amount of silica gel: the amount of the aqueous phase = 3:1, and the sample is dried under reduced pressure to obtain 51.2g of the yellow-green curly hair mushroom water phase mixed sample; wherein the conditions for reduced pressure drying are: vacuum degree 50mbar, temperature 40°C;

[0052] Step 2, QuikSep PS medium-pressure chromatography crude fractionation: The yellow-green curly mushroom water-phase mixed sample is separated by medium-pressure chromatography equipped with QuikSep PS, and detected by an ultraviolet detector with a detection wavelength of 210 nm. Six main fractions are collected according to the chromatographic peak shape, and then each fraction is dried under reduced pressure to obtain a total of six component samples of Fr1, Fr2, Fr3, Fr4, Fr5, and Fr6; wherein, the reduced pressure drying conditions are: vacuum degree 50 mbar, temperature 40°C; the medium-pressure chromatography separation working parameters of the QuikSep PS column are: the chromatographic column size is 460×49 mm, the stationary phase is QuikSep PS, the mobile phase A is water, B is methanol, the chromatographic conditions are 0-150 minutes, 100% A, 150-500 minutes, 0-70% B; the injection volume is 51.2 g; the flow rate is 40 mL / min, and the detection wavelength is 210 nm;

[0053] Step 3, initial screening of anti-liver injury active components: 2.5 mg of each of the above 6 component samples were taken and prepared into a sample solution with a concentration of 5 mg / mL using DMSO; human liver cancer cells HepG2 were cultured at a concentration of 2.5×10 5The cells were inoculated in a 6-well plate at a density of 100 μg / mL. After the cells adhered to the wall and grew to the logarithmic growth phase, each component and oleic acid at a working concentration of 600 μM were added to the 6-well plate. The final concentration of each component was 1 mg / mL and incubated for 24 hours. After 24 hours, the culture medium was discarded, the cells were fixed with 4% formaldehyde for 15 minutes, the residual 4% formaldehyde was washed off with distilled water, the cells were rinsed with isopropanol for 20 seconds, and Oil Red O staining was performed for 40 minutes. After 40 minutes, the residual Oil Red O stain was washed off with distilled water. The cells were soaked in isopropanol for 40 minutes to dissolve the Oil Red O solution. The multifunctional microplate reader was turned on and preheated for 5 minutes. The absorbance of each component was detected at a wavelength of 510 nm to quantitatively assess the anti-liver injury activity of each component. Finally, it was determined that the anti-liver injury activity of component Fr5 was the best. Compared with the normal group, #### p<0.0001; compared with the model group, *** p<0.001, **** p<0.0001; the initial screening of the anti-liver injury active components was divided into: normal group, model group, and drug administration group (6 components). The normal group only added DMSO with the same concentration as the drug administration group, the model group only added oleic acid with a working concentration of 600μM, and the drug administration group was 6 components + DMSO + 600μM oleic acid; other conditions remained the same;

[0054] Step 4, preparation of Megress C18 reverse phase preparative column: 372.7 mg of component Fr5 is added to 5 times its volume of methanol for dissolution, and then 2 times its volume of silica gel is added to mix and dry under reduced pressure to obtain 746 mg of Fr5 mixed sample; the Fr5 mixed sample is separated on Megress C18 preparative chromatography, and the recovered components are numbered and dried under reduced pressure to obtain 15 component samples including Fr5-1, Fr5-2, Fr5-3, Fr5-4, Fr5-5, Fr5-6, Fr5-7, Fr5-8, Fr5-9, Fr5-10, Fr5-11, Fr5-12, Fr5-13, Fr5-14 and Fr5-15; wherein the conditions for reduced pressure drying are: vacuum degree 50 mbar, temperature 40°C; Megress The working parameters of high pressure chromatography separation of C18 column are as follows: column size 250×20 mm; mobile phase A is water, B is methanol, chromatographic conditions are 0-60 min, 0-40% B; flow rate is 19 mL / min, detection wavelength is 210 nm;

[0055] Step 5, rescreening of active components against liver damage: 2.5 mg of each of the 15 component samples was taken and prepared into a sample solution with a concentration of 5 mg / mL using DMSO; human liver cancer cells HepG2 were cultured at a concentration of 2.5×10 5The cells were inoculated in a 6-well plate at a density of 100 μg / mL. After the cells adhered to the wall and grew to the logarithmic growth phase, each component and oleic acid with a working concentration of 600 μM were added to the 6-well plate. The final concentration of each component was 1 mg / mL and incubated for 24 hours. After 24 hours, the culture medium was discarded, the cells were fixed with 4% formaldehyde for 15 minutes, the residual 4% formaldehyde was washed off with distilled water, and the cells were rinsed with isopropanol for 20 seconds. Oil Red O staining was performed for 40 minutes. After 40 minutes, the residual Oil Red O stain was washed off with distilled water. The cells were soaked in isopropanol for 40 minutes to dissolve the Oil Red O solution. The multifunctional microplate reader was turned on and preheated for 5 minutes. The absorbance of each component was detected at a wavelength of 510 nm to quantitatively evaluate the anti-liver injury activity of each component. Finally, it was determined that the component Fr5-9 had the best anti-liver injury activity. Compared with the normal group, #### p<0.0001; compared with the model group, *** p<0.001, **** p<0.0001; Among them, the anti-liver injury active components were further screened into: normal group, model group, and drug administration group (15 components). The normal group only added DMSO with the same concentration as the drug administration group, the model group only added oleic acid with a working concentration of 600μM, and the drug administration group was 15 components + DMSO + 600μM oleic acid; other conditions remained the same;

[0056] Step 6, preparation of ACE Excel C18 AR reverse phase preparative column: add 5 times the volume of methanol and water to dissolve the component Fr5-9, the volume ratio of methanol to water is 2:3, then add 2 times the volume of silica gel to mix and dry under reduced pressure to obtain 86.5 mg of Fr5-9 mixed sample; separate the Fr5-9 mixed sample on ACE Excel C18 AR preparative chromatography and dry under reduced pressure to obtain 4.3 mg of sample Fr5-9-3 with a purity greater than 95%, and its spectral information; wherein, the conditions for reduced pressure drying are: vacuum degree 50 mbar, temperature 40°C; the high pressure chromatography separation working parameters of ACE Excel C18AR column are: the size of the chromatographic column is 250×10 mm; mobile phase A is water, B is acetonitrile, the chromatographic conditions are 0 to 30 minutes, 8% B; the flow rate is 19 mL / min, and the detection wavelength is 210 nm;

[0057] Step 7, in vitro evaluation of anti-liver injury activity: The sample Fr5-9-3 was prepared into a sample solution with a concentration of 1 mM using DMSO; human liver cancer cells HepG2 were cultured at a concentration of 2.5×10 5The cells were inoculated in a 6-well plate at a density of 10 μM. After the cells adhered to the wall and grew to the logarithmic growth phase, the Fr5-9-3 sample solution and oleic acid with a working concentration of 600 μM were added to the 6-well plate. The final concentration of the Fr5-9-3 sample solution was 10 μM. The cells were incubated for 24 hours. After 24 hours, the culture medium was discarded, the cells were fixed with 4% formaldehyde for 15 minutes, the residual 4% formaldehyde was washed off with distilled water, and the cells were rinsed with isopropanol for 20 seconds. The cells were stained with Oil Red O for 40 minutes, and the residual Oil Red O dye was washed off with distilled water after 40 minutes. The cells were soaked in isopropanol for 40 minutes to dissolve the Oil Red O solution. The multifunctional microplate reader was turned on and preheated for 5 minutes. The absorbance was detected at a wavelength of 510 nm to determine that Fr5-9-3 had in vitro anti-liver injury activity. Compared with the normal group, #### p<0.0001; compared with the model group, ** p<0.01, and named as Flolutsin C, chemical structure of Flolutsin C; among them, the in vitro anti-liver injury activity evaluation was divided into: normal group, model group, and drug administration group (sample Fr5-9-3). The normal group only added DMSO with the same concentration as the drug administration group, the model group only added oleic acid with a working concentration of 600μM, and the drug administration group was sample Fr5-9-3+DMSO+600μM oleic acid; other conditions remained consistent.

[0058] The application of the new dipeptide Flolutsin C with anti-liver damage activity in the above-mentioned Pleurotus eryngii, wherein the new dipeptide Flolutsin C is used as an active ingredient in combination with any acceptable carrier to prepare a drug for anti-liver damage, for example, to prepare a drug for anti-nonalcoholic fatty liver disease.

[0059] Example 2

[0060] The separation process of the new dipeptide Flolutsin C with anti-liver damage activity from Pleurotus eryngii specifically comprises the following steps:

[0061] Step 1, extraction: take 500g of fresh yellow-green curly hair mushroom sample, roughly chop it, extract it with 95% ethanol at room temperature according to the solid-liquid ratio of 1g:50mL, extract it 3 times, each time for 18 hours, filter and combine the filtrate, that is, filtrate A, and reduce pressure and dry the filtrate A to a paste to obtain the yellow-green curly hair mushroom ethanol extract extract; the yellow-green curly hair mushroom ethanol extract extract is extracted with petroleum ether and water in a volume ratio of 1:1, the aqueous phase is concentrated, the sample is mixed according to the amount of silica gel: the amount of the aqueous phase = 3:1, and the sample is dried under reduced pressure to obtain 194.7g of the yellow-green curly hair mushroom water phase mixed sample; wherein the conditions for reduced pressure drying are: vacuum degree 150mbar, temperature 50°C;

[0062] Step 2, QuikSep PS medium-pressure chromatography crude fractionation: The yellow-green shimeji water-phase mixed sample is separated by medium-pressure chromatography equipped with QuikSep PS, and detected by an ultraviolet detector with a detection wavelength of 210 nm. Six main fractions are collected according to the chromatographic peak shape, and then each fraction is dried under reduced pressure to obtain a total of six component samples of Fr1, Fr2, Fr3, Fr4, Fr5, and Fr6; wherein, the reduced pressure drying conditions are: vacuum degree 150 mbar, temperature 50°C; the medium-pressure chromatography separation working parameters of the QuikSep PS column are: the chromatographic column size is 460×49 mm, the stationary phase is QuikSep PS, the mobile phase A is water, B is methanol, the chromatographic conditions are 0-150 minutes, 100% A, 150-500 minutes, 0-70% B; the injection volume is 51.2 g; the flow rate is 40 mL / min, and the detection wavelength is 210 nm;

[0063] Step 3, initial screening of anti-liver injury active components: 2.5 mg of each of the above 6 component samples were taken and prepared into a sample solution with a concentration of 5 mg / mL using DMSO; human liver cancer cells HepG2 were cultured at a concentration of 2.5×10 5 The cells were inoculated in a 6-well plate at a density of 100 μg / mL. After the cells adhered to the wall and grew to the logarithmic growth phase, each component and oleic acid at a working concentration of 600 μM were added to the 6-well plate. The final concentration of each component was 1 mg / mL and incubated for 24 hours. After 24 hours, the culture medium was discarded, the cells were fixed with 4% formaldehyde for 15 minutes, the residual 4% formaldehyde was washed off with distilled water, the cells were rinsed with isopropanol for 20 seconds, and Oil Red O staining was performed for 40 minutes. After 40 minutes, the residual Oil Red O stain was washed off with distilled water. The cells were soaked in isopropanol for 40 minutes to dissolve the Oil Red O solution. The multifunctional microplate reader was turned on and preheated for 5 minutes. The absorbance of each component was detected at a wavelength of 510 nm to quantitatively assess the anti-liver injury activity of each component. Finally, it was determined that the anti-liver injury activity of component Fr5 was the best. Compared with the normal group, #### p<0.0001; compared with the model group, *** p<0.001, **** p<0.0001; the initial screening of the anti-liver injury active components was divided into: normal group, model group, and drug administration group (6 components). The normal group only added DMSO with the same concentration as the drug administration group, the model group only added oleic acid with a working concentration of 600μM, and the drug administration group was 6 components + DMSO + 600μM oleic acid; other conditions remained the same;

[0064] Step 4, Megress C18 reverse phase preparation column preparation: 1.47 g of component Fr5 was dissolved in 5 times its volume of methanol, and then 2 times its volume of silica gel was added to mix and dried under reduced pressure to obtain 3.06 g of Fr5 mixed sample; the Fr5 mixed sample was subjected to Megress The components were separated on C18 preparative chromatography, and the recovered components were numbered and dried under reduced pressure to obtain 15 component samples, namely Fr5-1, Fr5-2, Fr5-3, Fr5-4, Fr5-5, Fr5-6, Fr5-7, Fr5-8, Fr5-9, Fr5-10, Fr5-11, Fr5-12, Fr5-13, Fr5-14, and Fr5-15; wherein the conditions for reduced pressure drying were: vacuum degree 150 mbar, temperature 50°C; the working parameters for high-pressure chromatography separation of Megress C18 column were: column size 250×20 mm; mobile phase A was water, B was methanol, chromatographic conditions were 0 to 60 minutes, 0 to 40% B; flow rate was 19 mL / min, and detection wavelength was 210 nm;

[0065] Step 5, rescreening of active components against liver damage: 2.5 mg of each of the 15 component samples was taken and prepared into a sample solution with a concentration of 5 mg / mL using DMSO; human liver cancer cells HepG2 were cultured at a concentration of 2.5×10 5 The cells were inoculated in a 6-well plate at a density of 100 μg / mL. After the cells adhered to the wall and grew to the logarithmic growth phase, each component and oleic acid with a working concentration of 600 μM were added to the 6-well plate. The final concentration of each component was 1 mg / mL and incubated for 24 hours. After 24 hours, the culture medium was discarded, the cells were fixed with 4% formaldehyde for 15 minutes, the residual 4% formaldehyde was washed off with distilled water, and the cells were rinsed with isopropanol for 20 seconds. Oil Red O staining was performed for 40 minutes. After 40 minutes, the residual Oil Red O stain was washed off with distilled water. The cells were soaked in isopropanol for 40 minutes to dissolve the Oil Red O solution. The multifunctional microplate reader was turned on and preheated for 5 minutes. The absorbance of each component was detected at a wavelength of 510 nm to quantitatively evaluate the anti-liver injury activity of each component. Finally, it was determined that the component Fr5-9 had the best anti-liver injury activity. Compared with the normal group, #### p<0.0001; compared with the model group, *** p<0.001, **** p<0.0001; Among them, the anti-liver injury active components were further screened into: normal group, model group, and drug administration group (15 components). The normal group only added DMSO with the same concentration as the drug administration group, the model group only added oleic acid with a working concentration of 600μM, and the drug administration group was 15 components + DMSO + 600μM oleic acid; other conditions remained the same;

[0066] Step 6, preparation of ACE Excel C18 AR reverse phase preparative column: add 5 times the volume of methanol and water to dissolve the component Fr5-9, the volume ratio of methanol to water is 2:3, then add 2 times the volume of silica gel to mix and dry under reduced pressure to obtain 734.6 mg of Fr5-9 mixed sample; separate the Fr5-9 mixed sample on ACE Excel C18 AR preparative chromatography and dry under reduced pressure to obtain 54.6 mg of sample Fr5-9-3 with a purity greater than 95%, and its spectral information; wherein, the conditions for reduced pressure drying are: vacuum degree 150 mbar, temperature 50°C; the high pressure chromatography separation working parameters of ACE Excel C18 AR column are: the size of the chromatographic column is 250×10 mm; mobile phase A is water, B is acetonitrile, the chromatographic conditions are 0 to 30 minutes, 8% B; the flow rate is 19 mL / min, and the detection wavelength is 210 nm;

[0067] Step 7, in vitro evaluation of anti-liver injury activity: The sample Fr5-9-3 was prepared into a sample solution with a concentration of 1 mM using DMSO; human liver cancer cells HepG2 were cultured at a concentration of 2.5×10 5 The cells were inoculated in a 6-well plate at a density of 10 μM. After the cells adhered to the wall and grew to the logarithmic growth phase, the Fr5-9-3 sample solution and oleic acid with a working concentration of 600 μM were added to the 6-well plate. The final concentration of the Fr5-9-3 sample solution was 10 μM. The cells were incubated for 24 hours. After 24 hours, the culture medium was discarded, the cells were fixed with 4% formaldehyde for 15 minutes, the residual 4% formaldehyde was washed off with distilled water, and the cells were rinsed with isopropanol for 20 seconds. The cells were stained with Oil Red O for 40 minutes, and the residual Oil Red O dye was washed off with distilled water after 40 minutes. The cells were soaked in isopropanol for 40 minutes to dissolve the Oil Red O solution. The multifunctional microplate reader was turned on and preheated for 5 minutes. The absorbance was detected at a wavelength of 510 nm to determine that Fr5-9-3 had in vitro anti-liver injury activity. Compared with the normal group, #### p<0.0001; compared with the model group, ** p<0.01, and named as Flolutsin C, chemical structure of Flolutsin C; among them, the in vitro anti-liver injury activity evaluation was divided into: normal group, model group, and drug administration group (sample Fr5-9-3). The normal group only added DMSO with the same concentration as the drug administration group, the model group only added oleic acid with a working concentration of 600μM, and the drug administration group was sample Fr5-9-3+DMSO+600μM oleic acid; other conditions remained consistent.

[0068] The application of the new dipeptide Flolutsin C with anti-liver damage activity in the above-mentioned Pleurotus eryngii, wherein the new dipeptide Flolutsin C is used as an active ingredient in combination with any acceptable carrier to prepare a drug for anti-liver damage, for example, to prepare a drug for anti-nonalcoholic fatty liver disease.

[0069] Example 3

[0070] The separation process of the new dipeptide Flolutsin C with anti-liver damage activity from Pleurotus eryngii specifically comprises the following steps:

[0071] Step 1, extraction: take 1000g of fresh yellow-green curly hair mushroom sample, roughly chop it, and extract it with 95% ethanol at room temperature according to the solid-liquid ratio of 1g:100mL, extract it 4 times, each time for 24 hours, filter and combine the filtrate, that is, filtrate A, and reduce pressure and dry the filtrate A to a paste to obtain the yellow-green curly hair mushroom ethanol extract extract; the yellow-green curly hair mushroom ethanol extract extract is extracted with petroleum ether and water in a volume ratio of 1:1, the aqueous phase is concentrated, and the sample is mixed according to the amount of silica gel: the amount of the aqueous phase = 3:1 and dried under reduced pressure to obtain 397.6g of the yellow-green curly hair mushroom water phase mixed sample; wherein the conditions for reduced pressure drying are: vacuum degree 250mbar, temperature 60°C;

[0072] Step 2, QuikSep PS medium pressure chromatography crude fractionation: The sample of the yellow green mushroom water phase mixture was separated by medium pressure chromatography equipped with QuikSep PS, and detected by a UV detector with a detection wavelength of 210nm. Six major fractions were collected according to the chromatographic peak shape (see attached for details). Figure 1 ), and then each fraction was dried under reduced pressure to obtain 6 component samples of Fr1, Fr2, Fr3, Fr4, Fr5, and Fr6; wherein the conditions for reduced pressure drying are: vacuum degree 250mbar, temperature 60°C; the working parameters of medium-pressure chromatography separation of QuikSep PS column are: the size of the chromatographic column is 460×49mm, the stationary phase is QuikSep PS, the mobile phase A is water, B is methanol, the chromatographic conditions are 0-150 minutes, 100% A, 150-500 minutes, 0-70% B; the injection volume is 51.2g; the flow rate is 40mL / min, and the detection wavelength is 210nm;

[0073] Step 3, initial screening of anti-liver injury active components: 2.5 mg of each of the above 6 component samples were taken and prepared into a sample solution with a concentration of 5 mg / mL using DMSO; human liver cancer cells HepG2 were cultured at a concentration of 2.5×10 5The cells were inoculated in 6-well plates at a density of 100 μg / mL. After the cells adhered to the wall and grew to the logarithmic growth phase, each component and oleic acid with a working concentration of 600 μM were added to the 6-well plates. The final concentration of each component was 1 mg / mL and incubated for 24 hours. After 24 hours, the culture medium was discarded, the cells were fixed with 4% formaldehyde for 15 minutes, the residual 4% formaldehyde was washed off with distilled water, the cells were rinsed with isopropanol for 20 seconds, and Oil Red O staining was performed for 40 minutes. After 40 minutes, the residual Oil Red O stain was washed off with distilled water. The cells were soaked in isopropanol for 40 minutes to dissolve the Oil Red O solution. The multifunctional microplate reader was turned on and preheated for 5 minutes. The absorbance of each component was detected at a wavelength of 510 nm to quantitatively evaluate the anti-liver injury activity of each component. Finally, it was determined that the component Fr5 had the best anti-liver injury activity (see Appendix for details). Figure 2 ), compared with the normal group, #### p<0.0001; compared with the model group, *** p<0.001, **** p<0.0001; the initial screening of the anti-liver injury active components was divided into: normal group, model group, and drug administration group (6 components). The normal group only added DMSO with the same concentration as the drug administration group, the model group only added oleic acid with a working concentration of 600μM, and the drug administration group was 6 components + DMSO + 600μM oleic acid; other conditions remained the same;

[0074] Step 4, Megress C18 reverse phase preparative column preparation: 3.2 g of component Fr5 was dissolved in 5 times its volume of methanol, and then 2 times its volume of silica gel was added to mix and dried under reduced pressure to obtain 7.6 g of Fr5 mixed sample; the Fr5 mixed sample was separated by Megress C18 preparative chromatography, and the recovered components were numbered and dried under reduced pressure to obtain 15 component samples including Fr5-1, Fr5-2, Fr5-3, Fr5-4, Fr5-5, Fr5-6, Fr5-7, Fr5-8, Fr5-9, Fr5-10, Fr5-11, Fr5-12, Fr5-13, Fr5-14 and Fr5-15 (see attached for details). Figure 3 ); wherein the conditions for reduced pressure drying are: vacuum degree 250 mbar, temperature 60°C; the working parameters for high pressure chromatography separation of Megress C18 column are: column size 250×20 mm; mobile phase A is water, B is methanol, chromatographic conditions are 0 to 60 minutes, 0 to 40% B; flow rate is 19 mL / min, and detection wavelength is 210 nm;

[0075] Step 5, rescreening of active components against liver damage: 2.5 mg of each of the 15 component samples was taken and prepared into a sample solution with a concentration of 5 mg / mL using DMSO; human liver cancer cells HepG2 were cultured at a concentration of 2.5×10 5The cells were inoculated in a 6-well plate at a density of 100 μg / mL. After the cells adhered to the wall and grew to the logarithmic growth phase, each component and oleic acid with a working concentration of 600 μM were added to the 6-well plate. The final concentration of each component was 1 mg / mL and incubated for 24 hours. After 24 hours, the culture medium was discarded, the cells were fixed with 4% formaldehyde for 15 minutes, the residual 4% formaldehyde was washed off with distilled water, and the cells were rinsed with isopropanol for 20 seconds. Oil Red O staining was performed for 40 minutes. After 40 minutes, the residual Oil Red O stain was washed off with distilled water. The cells were soaked in isopropanol for 40 minutes to dissolve the Oil Red O solution. The multifunctional microplate reader was turned on and preheated for 5 minutes. The absorbance of each component was detected at a wavelength of 510 nm to quantitatively evaluate the anti-liver injury activity of each component. Finally, it was determined that the component Fr5-9 had the best anti-liver injury activity. Compared with the normal group, #### p<0.0001; compared with the model group, *** p<0.001, **** p<0.0001 (see Appendix Figure 4 ); Among them, the anti-liver injury active components were further screened into: normal group, model group, and drug administration group (15 components). The normal group only added DMSO with the same concentration as the drug administration group, the model group only added oleic acid with a working concentration of 600 μM, and the drug administration group was 15 components + DMSO + 600 μM oleic acid; other conditions remained the same;

[0076] Step 6, preparation of ACE Excel C18 AR reverse phase preparative column: add 5 times the volume of methanol and water to dissolve the component Fr5-9, the volume ratio of methanol to water is 2:3, then add 2 times the volume of silica gel to mix and dry under reduced pressure to obtain 1.2 g of Fr5-9 mixed sample; separate the Fr5-9 mixed sample on ACE Excel C18 AR preparative chromatography (see attached for details). Figure 5 ) and dried under reduced pressure to obtain 109.6 mg of sample Fr5-9-3 with a purity greater than 95%. Its spectral information (see attached Figure 7 ~Attached Fig.17 ); wherein the conditions for reduced pressure drying are: vacuum degree 250 mbar, temperature 60°C; the working parameters for high pressure chromatography separation of ACE Excel C18 AR column are: the size of the chromatographic column is 250×10 mm; the mobile phase A is water, B is acetonitrile, the chromatographic conditions are 0 to 30 minutes, 8% B; the flow rate is 19 mL / min, and the detection wavelength is 210 nm;

[0077] Step 7, in vitro evaluation of anti-liver injury activity: The sample Fr5-9-3 was prepared into a sample solution with a concentration of 1 mM using DMSO; human liver cancer cells HepG2 were cultured at a concentration of 2.5×10 5The cells were inoculated in a 6-well plate at a density of 10 μM. After the cells adhered to the wall and grew to the logarithmic growth phase, the Fr5-9-3 sample solution and oleic acid with a working concentration of 600 μM were added to the 6-well plate. The final concentration of the Fr5-9-3 sample solution was 10 μM. The cells were incubated for 24 hours. After 24 hours, the culture medium was discarded, the cells were fixed with 4% formaldehyde for 15 minutes, the residual 4% formaldehyde was washed off with distilled water, and the cells were rinsed with isopropanol for 20 seconds. The cells were stained with Oil Red O for 40 minutes, and the residual Oil Red O dye was washed off with distilled water after 40 minutes. The cells were soaked in isopropanol for 40 minutes to dissolve the Oil Red O solution. The multifunctional microplate reader was turned on and preheated for 5 minutes. The absorbance was detected at a wavelength of 510 nm to determine that Fr5-9-3 had in vitro anti-liver injury activity. Compared with the normal group, #### p<0.0001; compared with the model group, ** p<0.01 (see Appendix Figure 6 ) and named it Flolutsin C. The chemical structure of Flolutsin C (see the attached Fig.18 ); Among them, the in vitro evaluation of anti-liver injury activity was divided into: normal group, model group, and drug administration group (sample Fr5-9-3). The normal group only added DMSO with the same concentration as the drug administration group, the model group only added oleic acid with a working concentration of 600μM, and the drug administration group was sample Fr5-9-3+DMSO+600μM oleic acid; other conditions remained the same.

[0078] The application of the new dipeptide Flolutsin C with anti-liver damage activity in the above-mentioned Pleurotus eryngii, wherein the new dipeptide Flolutsin C is used as an active ingredient in combination with any acceptable carrier to prepare a drug for anti-liver damage, for example, to prepare a drug for anti-nonalcoholic fatty liver disease.

[0079] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A process for separating a novel dipeptide Flolutsin C from Pleurotus eryngii with anti-liver damage activity, characterized in that: The specific steps include: Step 1, extraction: after the fresh sample of the yellow-green curly mushroom is roughly crushed, it is extracted with 95% ethanol at room temperature according to a solid-liquid ratio of 1g:5mL-100mL, and the extraction is performed 2-4 times, each time for 12-24 hours, and the filtrate is filtered and combined, that is, filtrate A, and the filtrate A is dried under reduced pressure to a paste to obtain the yellow-green curly mushroom ethanol extract extract; the yellow-green curly mushroom ethanol extract extract is extracted with petroleum ether and water in a volume ratio of 1:1, and the aqueous phase is concentrated. The sample is mixed according to the amount of silica gel: the amount of the aqueous phase = 3:1 and dried under reduced pressure to obtain the yellow-green curly mushroom water phase mixed sample; Step 2, QuikSep PS medium-pressure chromatography crude fractionation: The sample of the yellow-green curcuma water phase mixture is separated by medium-pressure chromatography equipped with QuikSepPS, and detected by an ultraviolet detector with a detection wavelength of 210nm. Six main fractions are collected according to the chromatographic peak shape, and then each fraction is dried under reduced pressure to obtain six component samples of Fr1, Fr2, Fr3, Fr4, Fr5, and Fr6; Step 3, initial screening of anti-liver injury active components: each of the above 6 component samples was prepared into a sample solution with a concentration of 5 mg / mL using DMSO; human liver cancer cells HepG2 were cultured at a concentration of 2.5×10 5 The cells were inoculated in a 6-well plate at a density of 100 μg / cm2 and grown to the logarithmic growth phase after adherence to the wall. Then, each component and oleic acid with a working concentration of 600 μM were added to the 6-well plate and incubated for 24 hours. After 24 hours, the culture medium was discarded, the cells were fixed with 4% formaldehyde for 15 minutes, the residual 4% formaldehyde was washed off with distilled water, the cells were rinsed with isopropanol for 20 seconds, and Oil Red O staining was performed for 40 minutes. After 40 minutes, the residual Oil Red O staining solution was washed off with distilled water. The cells were soaked in isopropanol for 40 minutes to dissolve the Oil Red O solution. The multifunctional microplate reader was turned on and preheated for 5 minutes. The absorbance of each component was detected at a wavelength of 510 nm, and the anti-liver injury activity of each component was quantitatively evaluated. Finally, it was determined that the component Fr5 had the best anti-liver injury activity. Step 4, preparation of Megress C18 reverse phase preparative column: add 5 times the volume of methanol to dissolve component Fr5, then add 2 times the volume of silica gel to mix and dry under reduced pressure to obtain Fr5 mixed sample; separate the Fr5 mixed sample by Megress C18 preparative chromatography, number the recovered components and dry under reduced pressure to obtain 15 component samples including Fr5-1, Fr5-2, Fr5-3, Fr5-4, Fr5-5, Fr5-6, Fr5-7, Fr5-8, Fr5-9, Fr5-10, Fr5-11, Fr5-12, Fr5-13, Fr5-14 and Fr5-15; Step 5, rescreening of anti-liver injury active components: each of the 15 component samples was prepared into a sample solution with a concentration of 5 mg / mL using DMSO; human liver cancer cells HepG2 were cultured at a concentration of 2.5×10 5 The cells were inoculated in a 6-well plate at a density of 100 μM. After the cells adhered to the wall and grew to the logarithmic growth phase, each component and oleic acid at a working concentration of 600 μM were added to the 6-well plate and incubated for 24 hours. After 24 hours, the culture medium was discarded, the cells were fixed with 4% formaldehyde for 15 minutes, the residual 4% formaldehyde was washed off with distilled water, and the cells were rinsed with isopropanol for 20 seconds. Oil Red O staining was performed for 40 minutes. After 40 minutes, the residual Oil Red O stain was washed off with distilled water. The cells were soaked in isopropanol for 40 minutes to dissolve the Oil Red O solution. The multifunctional microplate reader was turned on and preheated for 5 minutes. The absorbance of each component was detected at a wavelength of 510 nm, and the anti-liver injury activity of each component was quantitatively evaluated. Finally, it was determined that the component Fr5-9 had the best anti-liver injury activity. Step 6, preparation of ACE Excel C18AR reverse phase preparative column: add 5 times the volume of methanol and water to dissolve component Fr5-9, wherein the volume ratio of methanol to water is 2:3, then add 2 times the volume of silica gel to mix well and dry under reduced pressure to obtain Fr5-9 mixed sample; separate the Fr5-9 mixed sample on ACE Excel C18AR preparative chromatography and dry under reduced pressure to obtain sample Fr5-9-3; Step 7, in vitro evaluation of anti-liver injury activity: The sample Fr5-9-3 was prepared into a sample solution with a concentration of 1 mM using DMSO; human liver cancer cells HepG2 were cultured at a concentration of 2.5×10 5 The cells were inoculated in a 6-well plate at a density of 100 μg / cm2 and grown to the logarithmic growth phase after adherence to the wall. The Fr5-9-3 sample solution and oleic acid with a working concentration of 600 μM were added to the 6-well plate and incubated for 24 hours. After 24 hours, the culture medium was discarded, the cells were fixed with 4% formaldehyde for 15 minutes, the residual 4% formaldehyde was washed off with distilled water, and the cells were rinsed with isopropanol for 20 seconds. The cells were stained with Oil Red O for 40 minutes, and the residual Oil Red O dye was washed off with distilled water after 40 minutes. The cells were soaked in isopropanol for 40 minutes to dissolve the Oil Red O solution, the multifunctional microplate reader was turned on and preheated for 5 minutes, and the absorbance was detected at a wavelength of 510 nm. It was determined that Fr5-9-3 had in vitro anti-liver injury activity and was named Flolutsin C. The chemical structure of Flolutsin C is:

2. The separation process of the novel dipeptide Flolutsin C with anti-liver damage activity from Pleurotus eryngii according to claim 1, characterized in that: In steps 1, 2, 4 and 6, the conditions for reduced pressure drying are: vacuum degree 50-250 mbar, temperature 40-60°C.

3. The separation process of the novel dipeptide Flolutsin C with anti-liver damage activity from Pleurotus eryngii according to claim 1, characterized in that: In step 2, the medium-pressure chromatographic separation operating parameters of the QuikSep PS column are: the column size is 460×49 mm, the stationary phase is QuikSep PS, the mobile phase A is water, B is methanol, the chromatographic conditions are 0-150 minutes, 100% A, 150-500 minutes, 0-70% B; the flow rate is 40 mL / min, and the detection wavelength is 210 nm.

4. The separation process of the novel dipeptide Flolutsin C with anti-liver damage activity from Pleurotus eryngii according to claim 1, characterized in that: In step 3, the anti-liver injury active components were initially screened into: normal group, model group, and drug administration group. The normal group only added DMSO at the same concentration as the drug administration group, the model group only added oleic acid at a working concentration of 600 μM, and the drug administration group was 6 components + DMSO + 600 μM oleic acid; other conditions remained consistent.

5. The separation process of the novel dipeptide Flolutsin C with anti-liver damage activity from Pleurotus eryngii according to claim 1, characterized in that: In step 4, the high pressure chromatography separation working parameters of the Megress C18 column are: the column size is 250×20 mm; the mobile phase A is water, B is methanol, the chromatographic conditions are 0 to 60 minutes, 0 to 40% B; the flow rate is 19 mL / min, and the detection wavelength is 210 nm.

6. The separation process of the novel dipeptide Flolutsin C with anti-liver damage activity from Pleurotus eryngii according to claim 1, characterized in that: In step 5, the anti-liver injury active components are further screened into: normal group, model group, and drug administration group. The normal group only adds DMSO at the same concentration as the drug administration group, the model group only adds oleic acid at a working concentration of 600 μM, and the drug administration group is 15 components + DMSO + 600 μM oleic acid; other conditions remain the same.

7. The separation process of the novel dipeptide Flolutsin C with anti-liver damage activity from Pleurotus eryngii according to claim 1, characterized in that: In step 6, the high pressure chromatography separation working parameters of the ACE Excel C18AR column are: the size of the chromatographic column is 250×10 mm; the mobile phase A is water, B is acetonitrile, the chromatographic conditions are 0 to 30 minutes, 8% B; the flow rate is 19 mL / min, and the detection wavelength is 210 nm.

8. The separation process of the novel dipeptide Flolutsin C with anti-liver damage activity from Pleurotus eryngii according to claim 1, characterized in that: In step 7, the in vitro evaluation of anti-liver injury activity is divided into: normal group, model group, and drug administration group. The normal group only adds DMSO at the same concentration as the drug administration group, the model group only adds oleic acid at a working concentration of 600 μM, and the drug administration group is sample Fr5-9-3+DMSO+600 μM oleic acid; other conditions remain the same.

9. Use of the new dipeptide Flolutsin C having anti-liver damage activity isolated from Pleurotus eryngii according to any one of claims 1 to 8, characterized in that: The novel dipeptide Flolutsin C is used as an effective ingredient and combined with any acceptable carrier to prepare medicines for resisting liver damage.

10. The use according to claim 9, characterized in that: The new dipeptide Flolutsin C is used for preparing medicines for resisting non-alcoholic fatty liver disease.

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