A marine fungus fermentation extract and its application

Abstract

The present invention provides a marine fungus extract and its application, belonging to the field of marine biomedicine technology. The extraction method of the marine fungus extract of the present invention comprises adding dried marine fungus powder to ethanol containing erucamide and 1,4-diiodobenzene, heating and extracting, decompressing and concentrating, and then freeze-drying. The marine fungus extract of the present invention can be used to prepare medicines and health products for preventing and treating non-alcoholic fatty liver, protecting the liver and reducing liver cell fat.

Description

Technical Field

[0001] The present invention relates to the technical field of marine biomedicine, and in particular to a marine fungus extract and application thereof. Background Art

[0002] The ocean is rich in microbial resources. The living environment characteristics such as high salt, high pressure, low temperature, special light, and oligotrophy determine the special metabolic mode of marine microorganisms, and the structure of secondary metabolites is complex and diverse. Marine fungi refer to a type of fungi that grows and forms spores in the ocean, salt water or estuary environment. Most of them are epiphytic or symbiotic in marine animals and plants or driftwood. Marine fungi are widely available, low-cost, and have a variety of active substances. Anti-tumor, antioxidant, antiviral, and antibacterial are their main types of biological activity. They have made great contributions to solving the clinical problems of modern medicine and the economic losses in agriculture caused by crop yield reductions due to pests and diseases every year.

[0003] Schizochytrium Schizochytriumlimacinum ) is a marine fungus with a high DHA content, belonging to the Thraustochytriaceae family. Schizochytrium is a single-celled, spherical fungus that accumulates a large amount of oil in its cells, accounting for more than 50% of the cell dry weight. More than 90% of the oil in the cells is easily absorbed by the human body, making it an ideal DHA resource. In addition, the polysaccharides extracted from Schizochytrium have biological activities such as antioxidant, antibacterial, lipid-lowering, and anti-tumor activities. Schizochytrium is cultured in a heterotrophic manner and grows fast.

[0004] Schizochytrium has become an ideal strain for fermentation production of DHA because its growth is not affected by seasons, it has many advantages such as no pollution to the environment and stable nutrients. Therefore, the research on Schizochytrium mainly focuses on the extraction and separation of DHA and the exploration of the activity of extracted polysaccharides, and there are few reports on the analysis and application of other nutrients of Schizochytrium. In addition, most of the research on Schizochytrium fermentation focuses on increasing the production of DHA, and there is little research on other extracts of Schizochytrium. Summary of the invention

[0005] The purpose of the present invention is to provide a marine fungus extract and an extraction method thereof which can be used to prepare medicines and health products for preventing and treating non-alcoholic fatty liver, protecting the liver and reducing liver cell fat; another purpose of the present invention is to provide a culture medium that can promote the growth and reproduction of marine fungi.

[0006] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical scheme.

[0007] A method for extracting a marine fungus extract comprises extracting with ethanol containing erucamide and 1,4-diiodobenzene.

[0008] Preferably, the weight ratio of erucamide to 1,4-diiodobenzene is 1-3:5-8.

[0009] Preferably, the marine fungus is Schizochytrium.

[0010] More preferably, the method for preparing the Schizochytrium extract comprises:

[0011] The Schizochytrium powder is added to ethanol containing erucamide and 1,4-diiodobenzene, and the mixture is heated for extraction, concentrated under reduced pressure, and freeze-dried to obtain the Schizochytrium extract.

[0012] More preferably, the method for preparing the Schizochytrium extract comprises:

[0013] The schizochytrium extract is obtained by adding 1-3 parts of erucamide, 5-8 parts of 1,4-diiodobenzene and 7-10 parts of 65-80% ethanol by weight to 1-3 parts of dried and crushed schizochytrium powder, heating to 70-80° C. in a water bath for extraction for 3-6 hours, concentrating under reduced pressure and freeze-drying.

[0014] The Schizochytrium extract extracted by the above method has the effect of protecting the liver, especially reducing the fat content of liver cells and reducing the AST and ALT contents in the liver, thereby playing a preventive and therapeutic role on non-alcoholic fatty liver disease.

[0015] Preferably, the Schizochytrium is cultured from a seed culture medium and a fermentation culture medium.

[0016] Preferably, the components of the seed culture medium include:

[0017] Glucose, monosodium glutamate, yeast extract, Na2SO4, K2SO4, Mg S O4·7H2O.

[0018] More preferably, the components of the seed culture medium include:

[0019] 60-70g / L glucose, 15-20g / L sodium glutamate, 12-18g / L yeast extract, 3-8g / L Na2SO4, 1-3g / L K2SO4, 0.5-0.8g / L MgSO4·7H2O;

[0020] Preferably, the components of the fermentation medium include:

[0021] Glucose, monosodium glutamate, yeast extract, Na2SO4, K2SO4, Mg S O4·7H2O, NaNO3, NH4SO4, vitamin B1, vitamin B6, biotin.

[0022] More preferably, the components of the fermentation medium include:

[0023] 60-70g / L glucose, 15-20g / L sodium glutamate, 12-18g / L yeast extract, 3-8g / L Na2SO4, 1-3g / L K2SO4, 0.5-0.8g / L MgSO4·7H2O, 2-5g / L NaNO3, 1-2g / L NH4SO4, 1-5mg / L vitamin B1, 10-20mg / L vitamin B6, 0.5-1mg / L biotin.

[0024] Schizochytrium is a marine fungus. In the traditional culture process, in order to simulate the seawater environment, a large amount of artificial sea salt needs to be added to the culture medium. In large-scale production, the presence of high-concentration sodium chloride easily leads to corrosion of chloride ions on the fermentation tank, increasing production costs. Therefore, in the present invention, sodium sulfate is used instead of sodium chloride, and substances containing chloride ions are avoided in other ingredients, which can provide necessary elements for the growth and reproduction of Schizochytrium and avoid damage to the fermentation equipment by chloride ions.

[0025] Preferably, the components of the fermentation medium also include: elaeuric acid and geniposide acid.

[0026] More preferably, the weight ratio of elaeuric acid to geniposide acid is 0.5-3:5-6.

[0027] More preferably, the components of the fermentation medium include:

[0028] 60-70g / L glucose, 15-20g / L sodium glutamate, 12-18g / L yeast extract, 3-8g / L Na2SO4, 1-3g / L K2SO4, 0.5-0.8g / L MgSO4·7H2O, 2-5g / L NaNO3, 1-2g / L NH4SO4, 0.5-3g elaeuric acid, 5-6g geniposide acid, 1-5mg / L vitamin B1, 10-20mg / L vitamin B6, 0.5-1mg / L biotin.

[0029] Adding elaeuric acid and geniposide acid to the culture medium can promote the growth and reproduction of Schizochytrium and increase the biomass of the fermentation broth in a short time; in addition, when using the above method to prepare Schizochytrium extract, the ethanol extract extracted from Schizochytrium cultured in a culture medium containing elaeuric acid and geniposide acid has better biological activity and a more obvious effect on reducing the fat content of liver cells.

[0030] Preferably, the method for culturing Schizochytrium comprises:

[0031] First use seed culture medium for cultivation and then transfer to fermentation medium for cultivation.

[0032] More preferably, the seed medium is used for culturing for 48-60 hours, and the fermentation medium is used for culturing for 2-5 days.

[0033] More preferably, the culture temperature using both culture media is 20-25°C.

[0034] More preferably, the method for culturing Schizochytrium comprises:

[0035] Pick a single colony of Schizochytrium and put it into 100-200 mL of seed culture medium, and culture it at 20-25° C. and 150-200 rpm for 48-60 hours; after the culture, inoculate the bacterial liquid into 50-100 mL of fermentation medium at a volume ratio of 8-10%, and culture it at 20-25° C. and 150-200 rpm for 2-5 days to obtain Schizochytrium fermentation liquid.

[0036] The invention also discloses the use of ethanol containing erucamide and 1,4-diiodobenzene for extraction.

[0037] The invention also discloses the use of the fermentation medium in promoting the growth and reproduction of marine fungi.

[0038] The invention also discloses the use of the extract in preparing a material for reducing liver AST and / or ALT index.

[0039] The invention also discloses the use of the extract in preparing medicine and / or health care product for reducing liver cell fat.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] The present invention uses ethanol containing erucamide and 1,4-diiodobenzene to extract the Schizochytrium extract, and the obtained extract has the effect of reducing the fat content of liver cells and reducing the AST and ALT content of the liver, making it close to the normal liver level, so it can be used to prepare medicines or health products for preventing and treating non-alcoholic fatty liver. In addition, the culture medium used for fermenting Schizochytrium in the present invention does not contain chloride ions, which can effectively avoid the damage of chloride ions to the fermentation equipment during the fermentation process, which is beneficial to reduce the equipment cost during large-scale fermentation; the fermentation medium used in the present invention is also added with elaeuric acid and geniposide acid, which can greatly increase the growth and reproduction speed of Schizochytrium, so that the fermentation liquid can reach the required biomass in a short time, and the fermentation time can be reduced to almost half of the fermentation time in the prior art. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is a HE staining photo of normal rat liver tissue;

[0043] Figure 2 This is a HE staining photograph of rat liver tissue that was intragastrically administered with the Schizochytrium extract prepared in Example 7;

[0044] Figure 3 The HE staining photograph of the liver tissue of the rats gavaged with the Schizochytrium extract prepared in Comparative Example 3. DETAILED DESCRIPTION

[0045] Here, exemplary embodiments will be described in detail, and the embodiments described in the following exemplary embodiments do not represent all embodiments consistent with the present disclosure. Instead, they are only examples of methods consistent with some aspects of the present disclosure as detailed in the appended claims.

[0046] The experimental methods in the following examples, unless otherwise specified, are all conventional methods or conditions recommended by the manufacturer. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

[0047] The rat alanine aminotransferase (ALT) ELISA kit and rat aspartate aminotransferase (AST) ELISA kit used in the following test examples were purchased from Shenzhen Zike Biotechnology Co., Ltd.; the rats and mice used and the feed used were purchased from Shanghai Jiake Biotechnology Co., Ltd.

[0048] Example 1

[0049] Preparation of Schizochytrium extract

[0050] 1. Cultivation of Schizochytrium

[0051] A single colony of Schizochytrium was picked and placed in 100 mL of seed culture medium, and cultured at 24°C with shaking at 200 rpm for 48 h; after the culture, the bacterial liquid was inoculated into 100 mL of fermentation medium at a volume ratio of 10%, and cultured at 24°C with shaking at 200 rpm for 4 days to obtain Schizochytrium fermentation liquid; the fermentation liquid was filtered, freeze-dried and crushed to obtain Schizochytrium powder;

[0052] The seed culture medium includes 65 g / L glucose, 18 g / L sodium glutamate, 12 g / L yeast extract, 6 g / L Na2SO4, 2 g / L K2SO4, and 0.8 g / L MgSO4·7H2O;

[0053] The fermentation medium includes: 65 g / L glucose, 18 g / L sodium glutamate, 12 g / L yeast extract, 6 g / L Na2SO4, 2 g / L K2SO4, 0.8 g / L MgSO4·7H2O, 3 g / L NaNO3, 1.2 g / L NH4SO4, 2 mg / L vitamin B1, 16 mg / L vitamin B6, and 0.8 mg / L biotin.

[0054] 2. Extraction of fermentation extract

[0055] Take 2 g of dried and crushed Schizochytrium powder, add 1.5 g of erucamide, 6 g of 1,4-diiodobenzene, and 10 g of 75% ethanol by volume, heat to 80°C in a water bath, extract for 4.5 h, then concentrate under reduced pressure and freeze-dry to obtain the Schizochytrium extract.

[0056] Example 2

[0057] Preparation of Schizochytrium extract

[0058] 1. Cultivation of Schizochytrium

[0059] A single colony of Schizochytrium was picked and placed in 100 mL of seed culture medium, and cultured at 24°C with shaking at 200 rpm for 48 h; after the culture, the bacterial liquid was inoculated into 100 mL of fermentation medium at a volume ratio of 10%, and cultured at 24°C with shaking at 200 rpm for 2 days to obtain Schizochytrium fermentation liquid; the fermentation liquid was filtered, freeze-dried and crushed to obtain Schizochytrium powder;

[0060] The seed culture medium includes 63 g / L glucose, 16 g / L sodium glutamate, 15 g / L yeast extract, 4 g / L Na2SO4, 1.8 g / L K2SO4, and 0.7 g / L MgSO4·7H2O;

[0061] The fermentation medium includes: 63 g / L glucose, 16 g / L sodium glutamate, 15 g / L yeast extract, 4 g / L Na2SO4, 1.8 g / L K2SO4, 0.7 g / L MgSO4·7H2O, 3 g / L NaNO3, 1.2 g / L NH4SO4, 1 g elaeuric acid, 3 g geniposidic acid, 2 mg / L vitamin B1, 16 mg / L vitamin B6, and 0.8 mg / L biotin.

[0062] 2. Extraction of fermentation extract

[0063] Same as Example 1.

[0064] Example 3

[0065] Preparation of Schizochytrium extract

[0066] 1. Cultivation of Schizochytrium

[0067] A single colony of Schizochytrium was selected and placed in 100 mL of seed culture medium, and cultured at 20°C with shaking at 150 rpm for 60 h; after the culture, the bacterial liquid was inoculated into 50 mL of fermentation culture medium at a volume ratio of 8%, and cultured at 20°C with shaking at 150 rpm for 2 days to obtain Schizochytrium fermentation liquid; the fermentation liquid was filtered, freeze-dried and crushed to obtain Schizochytrium powder;

[0068] The seed culture medium includes 60 g / L glucose, 15 g / L sodium glutamate, 12 g / L yeast extract, 3 g / L Na2SO4, 1 g / L K2SO4, and 0.5 g / L MgSO4·7H2O;

[0069] The fermentation medium includes: 60g / L glucose, 15g / L sodium glutamate, 12g / L yeast extract, 3g / L Na2SO4, 1g / L K2SO4, 0.5g / L MgSO4·7H2O, 2g / L NaNO3, 1g / L NH4SO4, 0.5g eleostearic acid, 5g geniposidic acid, 1mg / L vitamin B1, 10mg / L vitamin B6, and 0.5mg / L biotin.

[0070] 2. Extraction of fermentation extract

[0071] Same as Example 1.

[0072] Example 4

[0073] Preparation of Schizochytrium extract

[0074] 1. Cultivation of Schizochytrium

[0075] A single colony of Schizochytrium was picked and placed in 200 mL of seed culture medium, and cultured at 25°C with shaking at 200 rpm for 48 h; after the culture, the bacterial liquid was inoculated into 100 mL of fermentation culture medium at a volume ratio of 10%, and cultured at 25°C with shaking at 200 rpm for 2 days to obtain Schizochytrium fermentation liquid; the fermentation liquid was filtered, freeze-dried and crushed to obtain Schizochytrium powder;

[0076] The seed culture medium includes 70 g / L glucose, 20 g / L sodium glutamate, 18 g / L yeast extract, 8 g / L Na2SO4, 3 g / L K2SO4, and 0.8 g / L MgSO4·7H2O;

[0077] The fermentation medium includes: 70g / L glucose, 20g / L sodium glutamate, 18g / L yeast extract, 8g / LNa2SO4, 3g / L K2SO4, 0.8g / L MgSO4·7H2O, 5g / L NaNO3, 1-2g / L NH4SO4, 3g elaeuric acid, 6g geniposide acid, 5mg / L vitamin B1, 20mg / L vitamin B6, and 1mg / L biotin.

[0078] 2. Extraction of fermentation extract

[0079] Same as Example 1.

[0080] Example 5

[0081] Preparation of Schizochytrium extract

[0082] 1. Cultivation of Schizochytrium

[0083] The culture method and seed culture medium of this example are the same as those of Example 1. The difference between the fermentation culture medium and Example 1 is that it also contains 3 g of elaeuric acid. The culture time of the fermentation culture medium is 2 days.

[0084] 2. Extraction of fermentation extract

[0085] Same as Example 1.

[0086] Example 6

[0087] Preparation of Schizochytrium extract

[0088] 1. Cultivation of Schizochytrium

[0089] The culture method and seed culture medium of this example are the same as those of Example 1. The difference between the fermentation culture medium and Example 1 is that it also contains 6 g of geniposide acid. The culture time of the fermentation culture medium is 2 days.

[0090] 2. Extraction of fermentation extract

[0091] Same as Example 1.

[0092] Example 7

[0093] Preparation of Schizochytrium extract

[0094] 1. Cultivation of Schizochytrium

[0095] The culture method and seed culture medium of this example are the same as those of Example 1. The difference between the fermentation culture medium and Example 1 is that it also contains 3 g of elaeuric acid and 6 g of geniposidic acid. The culture time of the fermentation culture medium is 2 days.

[0096] 2. Extraction of fermentation extract

[0097] Same as Example 1.

[0098] Example 8

[0099] Preparation of Schizochytrium extract

[0100] 1. Cultivation of Schizochytrium

[0101] The difference between this example and Example 1 is that the culture time using the fermentation medium is 2 days.

[0102] 2. Extraction of fermentation extract

[0103] Same as Example 1.

[0104] Comparative Example 1

[0105] Preparation of Schizochytrium extract

[0106] 1. Cultivation of Schizochytrium

[0107] Same as Example 1.

[0108] 2. Extraction of fermentation extract

[0109] The difference between the extraction method of this comparative example and that of Example 1 is that 1,4-diiodobenzene is not added.

[0110] Comparative Example 2

[0111] Preparation of Schizochytrium extract

[0112] 1. Cultivation of Schizochytrium

[0113] Same as Example 1.

[0114] 2. Extraction of fermentation extract

[0115] The difference between the extraction method of this comparative example and that of Example 1 is that erucamide is not added.

[0116] Comparative Example 3

[0117] Preparation of Schizochytrium extract

[0118] 1. Cultivation of Schizochytrium

[0119] Same as Example 1.

[0120] 2. Extraction of fermentation extract

[0121] The difference between the extraction method of this comparative example and that of Example 1 is that erucamide and 1,4-diiodobenzene are not added.

[0122] Comparative Example 4

[0123] Preparation of Schizochytrium extract

[0124] 1. Cultivation of Schizochytrium

[0125] Same as Example 2.

[0126] 2. Extraction of fermentation extract

[0127] Same as Comparative Example 1.

[0128] Comparative Example 5

[0129] Preparation of Schizochytrium extract

[0130] 1. Cultivation of Schizochytrium

[0131] Same as Example 2.

[0132] 2. Extraction of fermentation extract

[0133] Same as Comparative Example 2.

[0134] Comparative Example 6

[0135] Preparation of Schizochytrium extract

[0136] 1. Cultivation of Schizochytrium

[0137] Same as Example 2.

[0138] 2. Extraction of fermentation extract

[0139] Same as Comparative Example 3.

[0140] Test Example 1

[0141] Fermentation broth biomass determination

[0142] The biomass of the fermentation broth in Examples 1-8 was determined as follows:

[0143] Take 30 mL of fermentation broth, centrifuge at 5000 rpm for 15 min, remove the supernatant, wash with distilled water and centrifuge again; dry and weigh the precipitate to calculate the biomass:

[0144] Biomass (g / L) = dry weight of bacteria (g) × 1000 / fermentation volume (mL).

[0145] The calculation results are shown in Table 1.

[0146] Table 1 Fermentation broth biomass

[0147] Biomass (g / L) Example 1 37.43 Example 2 37.68 Example 3 38.18 Example 4 37.91 Example 5 18.72 Example 6 17.49 Example 7 38.25 Example 8 18.51

[0148] As can be seen from Table 1, when the fermentation time is the same, the biomass of the fermentation broth in Examples 2-4 is not much different, all of which are above 35 g / L. The fermentation broth in Example 1 requires a longer fermentation time to reach a biomass close to that of Examples 2-4; the biomass of the fermentation broth in Examples 3 and 7 is close to and higher than that of other examples, reaching more than 38 g / L; when the amounts of the components of the culture medium are the same, the biomass of the fermentation broth in Examples 5, 6, and 8 is not much different, but is much smaller than the biomass in Example 7; This shows that the culture medium containing elaeuric acid and geniposide acid can accelerate the growth and reproduction of Schizochytrium, greatly increase the biomass in the fermentation broth in a short time, and shorten the fermentation time by about half.

[0149] Test Example 2

[0150] Functional assay of Schizochytrium extract

[0151] 1. Fatty liver rat model

[0152] After one week of adaptive feeding, SPF SD rats were divided into a normal group and a high-fat diet group. The high-fat diet group was further divided into a model group and a control group. The normal group was fed with normal feed, and the high-fat diet group was fed with high-fat feed for 4 consecutive weeks.

[0153] 2. Liver Sample Observation

[0154] The rats with fatty liver after modeling were randomly divided into 11 groups, with 5 rats in each group, groups 1-10 were sample groups, and group 11 was a control group; the Schizochytrium extracts in Examples 5-8 and Comparative Examples 1-6 were prepared into a suspension with distilled water, and then the mice were gavaged at a dose of 1000 mg / kg, once a day, for 2 weeks; the rats in the control group were gavaged with distilled water; after 2 weeks, one rat was randomly selected from each group, the rats were killed, the liver was fixed with glutaraldehyde, dehydrated step by step, embedded and sliced ​​with OTC, and observed after HE staining.

[0155] The results of the observations are as follows: Figure 1 As shown, the morphology and structure are normal, and there are no obvious fat vacuoles; the liver tissue staining results of rats gavaged with the Schizochytrium extract prepared in Example 7 are as shown Figure 2 As shown, its morphology and structure are similar to those of normal rat liver tissue, the cell cords are arranged neatly, the cell morphology and structure are normal, and there are few fat vacuoles; the results of liver tissue staining of rats gavaged with the Schizochytrium extract prepared in Comparative Example 3 are as follows Figure 3 As shown, more fat vesicles can be seen, the cell nucleus is squeezed and deformed, and the cell cord is broken. Figure 2 Similarly, the results of other comparative studies are similar to Figure 3 Similarly, no more photos are shown here.

[0156] The above results show that the Schizochytrium extract extracted in Example 7 has the effect of reducing liver cell fat, which is beneficial to the recovery of fatty liver to normal. It further shows that the Schizochytrium extract obtained by extraction with erucamide, 1,4-diiodobenzene and ethanol has the effect of reducing liver cell fat.

[0157] 3. Liver function index detection

[0158] The rats with fatty liver after modeling were randomly divided into 11 groups, with 5 rats in each group, groups 1-10 were sample groups, and group 11 was a control group; the Schizochytrium extracts in Examples 1, 5, 6, 7 and Comparative Examples 1-6 were prepared into a suspension with distilled water, and then the mice were gavaged at a dose of 1000 mg / kg, once a day, for 2 weeks; the rats in the control group were gavaged with distilled water; after 2 weeks, the rats were killed, the liver tissues were taken to prepare tissue homogenates, and the AST and ALT contents in the liver tissues were determined using a kit, and the determination results are shown in Table 2.

[0159] Table 2 Results of liver function tests in rats

[0160] AST content in liver tissue (U / L) ALT content in liver tissue (U / L) Example 1 102.48±1.25 24.53±2.17 Example 5 107.61±2.99 27.11±0.95 Example 6 105.82±4.68 26.53±1.93 Example 7 91.15±5.33 18.93±3.46 Comparative Example 1 168.21±2.33 50.66±1.73 Comparative Example 2 165.82±3.19 49.70±2.07 Comparative Example 3 172.79±4.21 52.04±2.44 Comparative Example 4 168.43±3.15 50.61±2.85 Comparative Example 5 171.20±3.43 51.22±1.93 Comparative Example 6 170.13±4.25 49.32±3.14 Control group 171.36±4.42 53.22±2.91 Normal mice 63.11±4.16 10.57±3.45

[0161] As can be seen from Table 2, by comparing the AST and ALT contents in the liver tissues of the sample group with those of the normal rats and the control group rats, it can be seen that the Schizochytrium extract prepared in Example 7 has a significant effect on reducing the AST and ALT contents in the liver tissue; the Schizochytrium extracts prepared in Examples 1, 5, and 6 also have the effect of reducing the AST and ALT contents in the liver tissue, but the effect is slightly worse than that of the Schizochytrium extract prepared in Example 7; and the Schizochytrium extract prepared in the comparative example hardly reduces the AST and ALT contents in the liver tissues of rats.

[0162] The above results indicate that only the Schizochytrium extract extracted using erucamide, 1,4-diiodobenzene and ethanol has the ability to reduce the activity of AST and ALT in liver tissue, and the fermented extract extracted from Schizochytrium cultured in a medium containing elaeuric acid and geniposidic acid has a stronger activity in reducing AST and ALT in liver tissue.

[0163] Test Example 3

[0164] Acute toxicity test

[0165] The Schizochytrium extracts prepared in Examples 1 and 7 were subjected to acute toxicity tests.

[0166] A total of 80 SPF Kunming mice were randomly divided into 10 groups, with 8 mice in each group, half of which were male and half were female. The experiment was conducted after one week of adaptive feeding: fasting but not water deprivation for 12 hours before gavage; distilled water was used to prepare the Schizochytrium extract suspension; the mice were gavaged at 1000mg / kg, 2000mg / kg, 3000mg / kg, 4000mg / kg, and 5000mg / kg; after gavage, the mice were observed for toxic reactions or death, and the observation was continued for 2 weeks.

[0167] The observation results are recorded in Table 3.

[0168] Table 3 Acute toxicity test results

[0169]

[0170] After gavage with the same dose of the Schizochytrium extract prepared in Example 1 and Example 7, the performance of the mice was similar: when the mice were gavaged with a dose of 1000 mg / kg and 2000 mg / kg, there were no other abnormal performances except that the mice showed a decrease in diet on the day after gavage; when the mice were gavaged with a dose of 3000 mg / kg, the mice showed mental depression and reduced activity in the first 3 days, which began to improve on the 4th to 7th days, and all aspects of the performance returned to normal after one week; when the mice were gavaged with a dose of 4000 mg / kg, the mice gradually showed hair loss and reduced diet on the 3rd to 5th days, and dead mice appeared on the 7th to 12th days; when the mice were gavaged with a dose of 3000 mg / kg, the mice gradually reduced their diet and showed uncoordinated movements in the first week, and all died within 10 days.

[0171] According to the results of the acute toxicity test, the median lethal dose of the Schizochytrium extract prepared in Example 1 was calculated by statistical software to be 3363.62 mg / kg; the median lethal dose of the Schizochytrium extract prepared in Example 7 was 3191.72 mg / kg; the median lethal doses of the two were close, indicating that culturing Schizochytrium using a culture medium containing elaeuric acid and geniposide acid would not produce toxic substances.

[0172] The conventional operations in the operating steps of the present invention are well known to those skilled in the art and will not be described in detail here.

[0173] The embodiments described above provide a detailed description of the technical solutions of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, supplements or similar substitutions made within the scope of the principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for extracting a marine fungus extract, characterized in that include:

1. Cultivation of Schizochytrium Pick a single colony of Schizochytrium and place it in 100-200 mL of seed culture medium: 60-70 g / L glucose, 15-20 g / L sodium glutamate, 12-18 g / L yeast extract, 3-8 g / L Na2SO4, 1-3 g / L K2SO4, 0.5-0.8 g / L MgSO4·7H2O, and culture at 20-25°C with shaking at 150-200 rpm for 48-60 hours; after the culture, inoculate the bacterial liquid into 50-100 mL of fermentation medium at a volume ratio of 8-10%: 60-70 g / L glucose, 15-20 g / L sodium glutamate, 12-18 g / L yeast extract, 3-8 g / L Na2SO4, 1-3 g / L K2SO4, 0.5-0.8 g / L MgSO4·7H2O, 2-5 g / L NaNO3, 1-2 g / L NH4SO4, 0.5-3g of elaeuric acid, 5-6g of geniposide, 1-5mg / L of vitamin B1, 10-20mg / L of vitamin B6, 0.5-1mg / L of biotin, culturing at 20-25°C and 150-200rpm for 2-5 days to obtain a schizochytrium fermentation liquid; filtering the fermentation liquid, freeze-drying and crushing it to obtain a schizochytrium powder; 2. Extraction of fermentation extract Take 1-3 parts of dried and crushed Schizochytrium powder, add 1-3 parts of erucamide, 5-8 parts of 1,4-diiodobenzene, and 7-10 parts of 65-80% ethanol by volume, heat to 70-80° C. in a water bath, extract for 3-6 hours, concentrate under reduced pressure, and then freeze-dry to obtain the Schizochytrium extract.

2. The marine fungus extract extracted by the extraction method of claim 1.

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