Monascus purpureus with anti-inflammatory immunocompetence and preparation method of monascus purpureus

Abstract

The invention relates to monascus with anti-inflammatory immunocompetence and a preparation method thereof, and belongs to the technical field of extraction and preparation of biological products. The monascus comprises monascus D, monascus E and monascus F, and is separated and prepared from a monascus liquid culture. The monascus is monascus purpureus (Monascus purpureus); the method comprises the following steps: performing liquid culture and domestication on monascus purpureus to obtain a monascus RCEF7056 strain, performing enlarged culture on the monascus RCEF7056 strain to obtain a liquid culture, and separating and purifying the liquid culture to obtain the monascus D, E and F. Preliminary activity analysis finds that the monascus D, E and F have very strong anti-inflammatory and immunosuppressive activity, and are expected to be used for treating chronic inflammation and developing related anti-aging products. The monascus is an edible fungus and is safe and reliable; the preparation method of the monascus D, E and F adopts microbial fermentation, is environment-friendly, is not influenced by natural environment and resources, and is easy to realize industrial, automatic and continuous production.

Description

Technical Field

[0001] The invention belongs to the technical field of extraction and preparation of biological products, and specifically relates to the fermentation, extraction, purification and structural identification of anti-inflammatory and immune Monascuskaolin D, Monascuskaolin E and Monascuskaolin F in a fermentation product of Monascus purpureus. Background Art

[0002] Monascus sp. is a small filamentous saprophytic fungus belonging to Eumycophyta, Ascomycotina, Plectomycetes, Eurotiales, Monascaceae, and Monascus. It includes Monascus purpureus, Monascus fuliginosus, and Monascus ruber. Monascus can produce a variety of enzymes such as saccharifying enzymes, esterifying enzymes, and proteases. Under the synergistic action of these enzymes, it can produce flavor substances such as monosaccharides, amino acids, and nucleotides, as well as aroma substances such as volatile acids, alcohols, and esters, making a significant contribution to the flavor of fermented foods.

[0003] However, the research on metabolites in red yeast rice started relatively late, especially the research on metabolites in liquid culture domesticated strains and their liquid fermentations, which seriously affects the safety of red yeast rice liquid fermented foods such as liquor, rice wine, cooking wine, fermented bean curd and sauce, and also does not utilize the development of related bioactive substances. Therefore, it is necessary to isolate and identify new compounds in its liquid culture.

[0004] Monascus RCEF7056 is a strain domesticated by long-term liquid fermentation. Preliminary analysis found that the red yeast liquid culture contains a large number of new bioactive substances that inhibit inflammation and immunity. Today's society is developing rapidly, with a fast pace of life and work and high pressure. At the same time, due to lack of exercise and bad habits such as staying up late to watch mobile phones, the number of sub-healthy people with immune disorders and chronic inflammation is increasing year by year. In particular, chronic inflammation can greatly reduce people's comfort and cause cancer. Therefore, it is necessary to develop anti-inflammatory immune-related drugs. As a food microorganism, Monascus has the advantages of safety and reliability, so related immune preparations have more application potential and huge economic value. Summary of the invention

[0005] The purpose of the present invention is to provide a red succinate with anti-inflammatory immune activity and a method for preparing the red succinate.

[0006] The present invention extracts monascus D, E and F from monascus RCEF7056 domesticated by red yeast liquid culture, and the monascus RCEF7056 is stored in the RCEF bacterial bank, namely the World Data Centre for Microorganisms, WDCM No. 1031 bacterial bank. The strain number is 7056.

[0007] A red purpurin with anti-inflammatory immune activity includes red purpurin D, red purpurin E and red purpurin F which are separated and prepared from the liquid culture of Monascus purpureus;

[0008] The monascusin D is a red powder with a molecular formula of C 25 H 31 NO 5 ; Monascus D has a rare N-ethanol group in its structure. The chemical name of Monascus D is:

[0009] The structural formula of E-7-(2-hydroxyethyl)-9a-methyl-3-octanoyl-6-(prop-1-en-1-yl)furo[3,2-g]isoquinoline-2,9(7H,9aH)-dione, red succinate D is as follows:

[0010]

[0011] The monascusin E is a yellow powder with a molecular formula of C 22 H 32 O 5 , the chemical name is (7R,8R)-7-hydroxy-3-(2-hydroxypropy)-7-methyl-8-(2-oxonony)-7,8-dihydro-6H-isochromen-6-one; the spatial structure of red yeast rice is as follows:

[0012]

[0013] The monascusin F is a yellow powder and is an isomer of monascusin E. Its chemical name is (7S,8S)-7-hydroxy-3-(2-hydroxypropy)-7-methyl-8-(2-oxonony)-7,8-dihydro-6H-isochromen-6-one. The spatial structural formula of monascusin F is as follows:

[0014]

[0015] The Monascus is Monascus purpureus; the Monascus RCEF7056 strain is obtained by liquid culture of Monascus purpureus, the liquid culture is obtained by expanding the culture of the Monascus RCEF7056 strain, and the liquid culture is separated and purified to obtain Monascus D, Monascus E and Monascus F;

[0016] 10 μM red yeast rice D reduced the transcription levels of IL-1β, TNF-α, and TGF-β in LPS-induced RAW264.7 cells by 81%, 75%, and 68%, respectively, which was stronger than the positive control dexamethasone (DEX) with the same concentration of anti-inflammatory immune activity;

[0017] 10 μM red yeast rice E reduced the transcription levels of IL-1β, TNF-α, and TGF-β in LPS-induced RAW264.7 cells by 67%, 59%, and 61%, respectively, which was close to the activity of the positive control dexamethasone (DEX) with anti-inflammatory immune activity at the same concentration;

[0018] 10 μM red yeast rice F reduced the transcription levels of IL-1β, TNF-α, and TGF-β in LPS-induced RAW264.7 cells by 77%, 86%, and 73%, respectively, which was stronger than the positive control dexamethasone (DEX) with anti-inflammatory immune activity at the same concentration.

[0019] The preparation steps of red yeast rice with anti-inflammatory immune activity are as follows:

[0020] (1) Culture (1.1) Dissolve the Monascus spores in 0.5% Tween 80 and then add sterile water to a concentration of 10 5～7 spores / mL of spore suspension, inoculated into 250mL of PDB liquid culture medium at 1-3% of the volume of each bottle, and cultured in a shaking bottle at a temperature of 32-35°C and a rotation speed of 220-250r / min for 7-10 days; repeating the cycle of inoculation and culture 5-9 times to obtain an acclimated liquid culture;

[0021] (1.2) inoculating the domesticated liquid culture onto a potato agar (PDA) medium, inoculating 200-1000 μL of the domesticated liquid culture into each culture dish of the potato agar (PDA) medium (11 cm in diameter), culturing at a temperature of 32-35° C. for 7-10 days, collecting spores, and obtaining liquid culture domesticated Monascus RCEF7056 strains;

[0022] (2) Preparation and expansion of liquid culture

[0023] The spore suspension of Monascus RCEF7056 is subjected to primary strain culture, secondary seed culture and tertiary liquid expansion culture to obtain an expanded liquid culture;

[0024] The primary bacterial culture is potato agar (PDA) slant culture;

[0025] The secondary seed culture is a liquid culture of PDB medium;

[0026] The tertiary liquid expansion culture is a liquid culture of PDB medium;

[0027] (3) Expanding the extraction and purification of active ingredients from liquid cultures

[0028] The extractant used for the extraction and purification is prepared by uniformly mixing methanol and ethyl acetate in a volume ratio of 0-1:1-0; the specific extraction and purification operations are as follows:

[0029] (3.1) Extraction of active ingredients from liquid culture

[0030] The expanded liquid culture is concentrated and dried under reduced pressure; ultrasonic extraction is performed using an extractant, membrane filtration or centrifugal separation is performed, and the solvent is removed by reduced pressure evaporation to obtain an effective component extract;

[0031] (3.2) Preliminary purification of active ingredient extract

[0032] Preliminary purification of the active ingredient extract was performed using preparative reverse-phase high performance liquid chromatography to obtain a red preliminary purified product and a yellow preliminary purified product;

[0033] (3.3) Purification of two preliminary purified products

[0034] The two preliminary purified products were purified separately by semi-preparative reversed-phase HPLC;

[0035] The red preliminary purified product was eluted with acetonitrile solution, and the compound corresponding to the highest chromatographic peak was collected as the monascus D eluate, and the solvent was evaporated to obtain red monascus D;

[0036] The yellow preliminary purified product was eluted with acetonitrile solution, and the eluents corresponding to the first highest chromatographic peak and the second highest chromatographic peak were collected, which were the eluent of Monascus E and the eluent of Monascus F, respectively. The solvent was evaporated to obtain yellow Monascus E and yellow Monascus F.

[0037] The further preparation technology scheme is as follows:

[0038] In step (2), the specific operations are as follows:

[0039] (2.1) Primary bacterial culture,

[0040] The spore suspension of Monascus RCEF7056 strain is 10 6The preparation method of the Monascus spore suspension is as follows: 0.1 g of spores are dissolved in 10 mL of 0.5% Tween 80, and then the spore concentration is diluted to 10 with sterile water. 6 spores / mL;

[0041] The spore suspension of Monascus RCEF7056 was inoculated on a potato agar (PDA) slant culture medium, with each slant inoculated with 10-30 μL of the spore suspension, and cultured at a temperature of 32-35° C. and 220-250 r / min for 7-9 days, and spores were collected to obtain a primary strain.

[0042] (2.2) Secondary seed culture

[0043] Dissolve the primary strain with 0.5% Tween 80 and dilute to 10% with sterile water. 5 ~10 7 spores / mL of spore suspension, inoculated into 250mL of liquid PDB medium at an inoculum volume of 200-250μL per bottle, and cultured in a shaking flask at a temperature of 32-35°C and a rotation speed of 220-250r / min for 3-5 days to obtain a secondary liquid strain;

[0044] (2.3) Tertiary liquid expansion culture

[0045] 1-3 mL of the secondary liquid bacterial strain was inoculated into 250 mL of liquid PDB medium, and cultured in a shaking flask at a temperature of 32-35° C. and a rotation speed of 220-250 r / min for 7-9 days to obtain a liquid culture.

[0046] In step (3.1), the specific operations are as follows:

[0047] The liquid culture is concentrated under reduced pressure to 3 / 10 to 1 / 10 of the original volume under the conditions of a vacuum degree of -0.1 to -0.08 MP and a temperature of 10 to 60°C; dried and crushed under the conditions of a temperature of -50 to 130°C, an extractant is added at a weight volume ratio of 1 g: 0.5 to 5 mL, and ultrasonic extraction is performed at 40 KHz for 20 to 200 minutes; centrifuged at 4000 to 15000 rpm, or filtered through a 0.1 to 2.5 μm membrane to obtain a filtrate or a centrifugal supernatant; the filtrate or the centrifugal supernatant is evaporated under reduced pressure to remove the extractant under the conditions of a vacuum degree of -0.1 to -0.08 MP and a temperature of 10 to 60°C to obtain an effective component extract.

[0048] In step (3.2), the specific operations are as follows:

[0049] The method adopts preparative reverse phase high performance liquid chromatography for purification; the chromatographic separation preparation conditions are as follows: mobile phase A is ultrapure water, and mobile phase B is acetonitrile; the elution conditions are as follows: 40% to 70% mobile phase B gradient elution from 0 to 30 minutes; the injection volume is 30 to 300 μL, the column temperature is 20 to 40° C., and the flow rate is 5 to 50 mL / min; the detection wavelength is 358 nm; the chromatographic column is a preparative reverse phase C-18 column; the red eluent and the yellow eluent are collected, and the extractant is evaporated under reduced pressure under the conditions of a vacuum degree of -0.1 to -0.08 MP and a temperature of 10 to 60° C., and the product is dried at a temperature of less than or equal to 130° C. to obtain a red preliminary purified product and a yellow preliminary purified product.

[0050] In step (3.3), the specific operations are as follows:

[0051] The chromatographic separation preparation conditions are as follows: mobile phase A is ultrapure water, mobile phase B is acetonitrile; elution conditions: elution with 40% to 70% mobile phase B from 0 to 30 min; injection volume 20 to 200 μL, column temperature 20 to 40° C., flow rate 2 to 20 mL / min; detection wavelength 358 nm; chromatographic column: semi-preparative reverse phase C-18 column;

[0052] Inject 20-200 mg / mL of the red preliminary purified product, collect the eluate corresponding to the highest chromatographic peak, and obtain the red chromatographic dye D eluate; remove the extractant by decompression evaporation of the eluate under the conditions of vacuum degree -0.1-0.08 MP and temperature 10-60° C., and dry at 130° C. or less; obtain the red solid red chromatographic dye D;

[0053] 20-200 mg / mL of the red preliminary purified product is injected, and the eluents corresponding to the first highest chromatographic peak and the second highest chromatographic peak are collected in sequence to obtain the eluents of Monascus E and Monascus F, respectively; these eluents are respectively evaporated under reduced pressure to remove the extractant under the conditions of a vacuum degree of -0.1 to -0.08 MP and a temperature of 10 to 60° C., and are dried at a temperature of less than or equal to 130° C.; yellow solid Monascus E and yellow solid Monascus F are respectively obtained.

[0054] In step (3.1), the extractant is methanol, or ethyl acetate, or a mixture of methanol and ethyl acetate.

[0055] The analytical research of the present invention is described as follows:

[0056] 1. The Monascus RCEF7056 strain used in the present invention is obtained by domesticating the common Monascus purpurogenum strain through repeated liquid culture.

[0057] 2. The study used preparative reversed-phase HPLC and semi-preparative reversed-phase HPLC methods to separate and purify the extracts. Three new compounds were separated and purified from the liquid fermentation product of Monascus RCEF7056 and named Monascus D, E and F.

[0058] 3. Chemical structure identification data of monascusin D

[0059] High-resolution liquid chromatography-mass spectrometry analysis showed that the mass-to-charge ratio of the ion of red succinate D was 426.1955, [M+H] + The corresponding molecular formula is C 25 H 31 NO 5 , the specific NMR data are shown in Table 1 below.

[0060] Table 1. NMR carbon and hydrogen assignments of red yeast rice D

[0061]

[0062]

[0063] 10 μM red polysaccharide D reduced the transcription levels of IL-1β, TNF-α, and TGF-β in LPS-induced RAW264.7 cells by 81%, 75%, and 68%, respectively, which was stronger than the positive control DEX with anti-inflammatory immune activity at the same concentration.

[0064] 4. Chemical structure identification data of Monascus E

[0065] High-resolution liquid chromatography-mass spectrometry analysis showed that the mass-to-charge ratio of the ion of red yeast rice was 377.2367, [M+H] + The corresponding molecular formula is C 22 H 32 O 5 , the specific NMR data are shown in Table 2 below.

[0066] Table 2. NMR carbon-hydrogen assignment table of red yeast rice

[0067]

[0068]

[0069] 10 μM red polysaccharide E reduced the transcription levels of IL-1β, TNF-α, and TGF-β in LPS-induced RAW264.7 cells by 67%, 59%, and 61%, respectively, which was close to the activity of the positive control DEX of anti-inflammatory immune activity at the same concentration.

[0070] 5. Spatial structure of red yeast rice

[0071] In order to explore the spatial structure of red succinate E, red succinate E was dissolved in chloroform solution. Figure 1 , and circular dichroism spectropolarimeter was performed. The results showed that the configuration of red succinate E is (7R, 8R), so its chemical name is:

[0072] (7R,8R)-7-hydroxy-3-(2-hydroxypropy)-7-methyl-8-(2-oxonony)-7,8-dihydro-6H-isochromen-6-one, the structural formula of red crotonin E is as follows:

[0073]

[0074] 6. Chemical structure identification data of Monascus F

[0075] High-resolution liquid chromatography-mass spectrometry analysis showed that the mass-to-charge ratio of the ion of red succinate F was 377.2367, [M+H] + The corresponding molecular formula is C 22 H 32 O 5 , which is the same as Monascusin E, indicating that Monascusin F is an isomer of E. The NMR data of Monascusin F are shown in Table 3.

[0076] Table 3. NMR carbon and hydrogen assignments of red yeast rice

[0077]

[0078]

[0079] 10 μM red yeast rice F reduced the transcription levels of IL-1β, TNF-α, and TGF-β in LPS-induced RAW264.7 cells by 77%, 86%, and 73%, respectively, which was stronger than the positive control DEX with anti-inflammatory immune activity at the same concentration.

[0080] 7. Spatial structure of red yeast rice F

[0081] Dissolve Monascusin F in methanol solution, see Figure 2 , circular dichroism spectroscopy (Circular Dichroism Spectropolarimeter), found that the configuration of red sulphate F is (7S, 8S), so its chemical name is:

[0082] (7S,8S)-7-hydroxy-3-(2-hydroxypropy)-7-methyl-8-(2-oxonony)-7,8-dihydro-6H-isochromen-6-one.

[0083] The structural formula of red yeast rice F is as follows:

[0084]

[0085] The beneficial technical effects of the present invention are embodied in the following aspects:

[0086] 1. In recent years, domestic and foreign scholars have discovered a large number of bioactive substances from red yeast rice, which have lipid-lowering, anti-oxidation, anti-cancer and antibacterial effects. Some have been developed into drugs, which shows that the fungus has great potential for utilization. Bioinformatics prediction shows that the genomic DNA of Monascus contains unknown gene clusters encoding polyketides and non-ribosomal peptides, which lays a potential foundation for the discovery of new secondary metabolites in Monascus. In order to find unreported new compounds in the metabolites of Monascus, the inventors first domesticated a large number of Monascus strains by liquid culture, and then extracted and analyzed the effective components of the culture by liquid chromatography-mass spectrometry, and determined that there are multiple new compounds in the metabolites of Monascus RCEF7056. Based on large-scale culture and extraction, reverse preparation and semi-preparative chromatography were used to separate and purify Monascus kaolin D, Monascus kaolin E and Monascus kaolin F, and the structural formulas of Monascus D, E and F were obtained by one-dimensional and two-dimensional nuclear magnetic resonance and accurate mass spectrometry. Preliminary activity analysis found that the red yeast rice D, E and F of the present invention have strong anti-inflammatory and immunosuppressive activities, and are expected to be used in the treatment of chronic inflammation and the development of related anti-aging products.

[0087] 2. The present invention found that Monascus RCEF7056 has the function of metabolizing the above substances. Based on this invention, it is expected to further clone related genes and construct high-yield strains.

[0088] 3. Since the present invention adopts microbial fermentation production, it is not affected by the natural environment and resources, and is easy to realize industrialized, automated, and continuous production without damaging the natural environment and natural resources. The red yeast rice product produced by the process method of the present invention has low cost, simple process, stable process, easy regulation, and high success rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0089] Figure 1 This is the circular dichroism spectrum of red succinate E.

[0090] Figure 2 This is the circular dichroism spectrum of red sulphur pigment F. DETAILED DESCRIPTION

[0091] The present invention will be further described below in conjunction with embodiments.

[0092] Example 1

[0093] The steps for extracting monascus D, E, and F from Monascus RCEF7056 are as follows:

[0094] (1) Strain domestication

[0095] The Monascus used was Monascus purpureus.

[0096] (1.1) Dissolve the Monascus spores in 0.5% Tween 80 to form a spore suspension, and then use sterile water to make a 10% 5 The spore suspension of spores / mL is inoculated into 250mL of PDB liquid culture medium at 1% of the volume of each bottle, and the culture is shaken for 7 days at a temperature of 32°C and a rotation speed of 220r / min; the first culture is inoculated into a newly prepared 220mL of PDB culture medium at an inoculation amount of 1-3% by volume, and the culture is shaken for 7 days at a temperature of 32°C and a rotation speed of 220r / min; the second culture is inoculated into a newly prepared 250mL of PDB culture medium at an inoculation amount of 1% by volume, and the culture is shaken for 7 days at a temperature of 32°C and a rotation speed of 220r / min. Then, the previous step is repeated, and the third culture is inoculated into a newly prepared 250mL of PDB culture medium at an inoculation amount of 1% by volume, and the culture is shaken for 7 days at a temperature of 32°C and a rotation speed of 220r / min. The inoculation culture is repeated for a total of 5 times to obtain an acclimated liquid culture.

[0097] (1.2) The domesticated liquid culture was inoculated onto potato agar (PDA) medium, with 2000 μL of the culture inoculated into each culture dish (11 cm in diameter), and cultured at 32° C. for 7 days. Spores were collected to obtain the domesticated Monascus RCEF7056 strain.

[0098] (2) Preparation and expansion of liquid culture

[0099] The Monascus purpurogenum RCEF7056 strain is subjected to primary strain culture, secondary seed culture and tertiary liquid expansion culture to obtain a liquid culture;

[0100] (2.1) Cultivation of primary bacterial strains

[0101] The spore suspension of Monascus RCEF7056 is 10 6 The preparation method of the Monascus spore suspension is as follows: 0.1 g of spores are dissolved in 10 mL of 0.5% Tween 80, and then the spore concentration is diluted to 10 with sterile water. 6spores / mL.

[0102] The spore suspension of Monascus RCEF7056 was inoculated on potato agar (PDA) slant culture medium, with 10 μL of the spore suspension inoculated on each slant, and cultured at 32° C. and 220 r / min for 7 days to obtain a primary strain.

[0103] PDA medium is potato dextrose medium, and its formula is: 200g peeled potatoes, 20g glucose and 15g agar, add water to 1000mL.

[0104] (2.2) Cultivating secondary seeds

[0105] Dissolve the primary strain in 0.5% Tween 80 and then make it into 10% with sterile water. 5 The spore suspension with a concentration of spores / mL was inoculated into 250 mL of liquid PDB medium at a rate of 200 μL per bottle, and the culture was carried out in a shaking flask at a temperature of 32° C. and a rotation speed of 220 r / min for 3 days to obtain a secondary liquid strain.

[0106] The formula of PDB medium is: 200 g peeled potato, 20 g glucose and water to 1000 mL.

[0107] (2.3) Tertiary liquid expansion culture

[0108] 1 mL of the secondary liquid strain was inoculated into 250 mL of liquid PDB medium, and cultured in a shaking flask at a temperature of 32° C. and a rotation speed of 220 r / min for 7 days, and the culture was collected to obtain a liquid culture.

[0109] (3) Extraction and purification of active ingredients from liquid cultures

[0110] The specific operations are as follows:

[0111] (3.1) Extraction of active ingredients from liquid culture

[0112] The culture was concentrated under reduced pressure and dried, and then extracted with ethyl acetate to obtain an effective component extract.

[0113] The specific operation is as follows: the liquid culture is evaporated to 3 / 10 of the original volume under vacuum degree -0.1MP and temperature 10℃, dried at temperature -50℃, and crushed; ethyl acetate is added as an extractant at a weight volume ratio of 1g:0.5mL, and ultrasonic extraction is performed at 40KHz for 20 minutes. Then centrifugation is performed at a speed of 4000 rpm, and the extractant is evaporated under vacuum degree -0.1MP and temperature 10℃ to obtain an effective component extract.

[0114] (3.2) Preliminary purification of active ingredient extracts

[0115] The active component extract was preliminarily purified by preparative reverse-phase high performance liquid chromatography to obtain a red preliminarily purified product and a yellow preliminarily purified product.

[0116] The specific operation is: using preparative reverse-phase high performance liquid chromatography for purification; the chromatographic separation preparation conditions are: mobile phase A is ultrapure water, and mobile phase B is acetonitrile; elution conditions: 0 to 30min, 40% to 70% mobile phase B gradient elution; injection volume 30μL, column temperature 20°C, flow rate 5mL / min; detection wavelength is 358nm; chromatographic column: preparative reverse-phase C-18 column; collecting the red eluent and the yellow eluent, under vacuum at -0.1MP and 10°C, the extractant is evaporated under reduced pressure, and dried at -40°C to obtain a red preliminary purified product and a yellow preliminary purified product.

[0117] (3.3) Purification of the Preliminary Purification Product

[0118] The two preliminary purified products were refined by semi-preparative reverse-phase high performance liquid chromatography. Under the elution condition of acetonitrile solution, the compounds corresponding to the chromatographic peaks were collected, which were the eluent of Monascus D, Monascus E and F. After evaporating and removing the solvent, red Monascus D and yellow Monascus E and F were obtained.

[0119] The chromatographic separation preparation conditions are as follows: chromatographic mobile phase A is ultrapure water, mobile phase B is acetonitrile; elution conditions: 0 to 30 min, elution with 40% to 70% mobile phase B; injection volume 20 μL, column temperature 20°C, flow rate 2 mL / min; detection wavelength 358 nm; chromatographic column: semi-preparative reverse phase C-18 column;

[0120] The specific operations are as follows:

[0121] Inject 20 mg / mL of the red preliminary purified product, collect the eluate corresponding to the highest chromatographic peak, and obtain the red chromatographic dye D eluate; remove the extractant from the eluate under reduced pressure at a vacuum degree of -0.1 MP and a temperature of 10°C, and dry at -45°C; obtain red solid red chromatographic dye D.

[0122] 20 mg / mL of the red preliminary purified product was injected, and the eluents corresponding to the first highest chromatographic peak and the second highest chromatographic peak were collected in turn to obtain the eluent of Monascus E and the eluent of Monascus F, respectively; the eluent of Monascus E and the eluent of Monascus F were evaporated under reduced pressure at a vacuum degree of -0.1 MP and a temperature of 10°C to remove the extractant, and then dried at -45°C; yellow solid Monascus E and yellow solid Monascus F were obtained, respectively.

[0123] Monascus succinate D is a red powder with a molecular formula of C 25 H 31 NO 510μM red yeast rice D reduced the transcription levels of IL-1β, TNF-α, and TGF-β in LPS-induced RAW264.7 cells by 81%, 75%, and 68%, respectively, which was stronger than the positive control DEX with the same concentration of anti-inflammatory immune activity. The structural formula of red yeast rice D is as follows:

[0124]

[0125] Monascus E is a yellow powder with a molecular formula of C 22 H 32 O 5 , chemical name is (7R,8R)-7-hydroxy-3-(2-hydroxypropy)-7-methyl-8-(2-oxonony)-7,8-dihydro-6H-isochromen-6-one. 10μM red yeast rice E reduced the transcription levels of IL-1β, TNF-α, and TGF-β in LPS-induced RAW264.7 cells by 67%, 59%, and 61%, respectively, which is close to the activity of the positive control DEX of the same concentration of anti-inflammatory immune activity.

[0126] The structural formula of red yeast rice is as follows:

[0127]

[0128] Monascusin F is a yellow powder with a chemical name of (7S,8S)-7-hydroxy-3-(2-hydroxypropy)-7-methyl-8-(2-oxonony)-7,8-dihydro-6H-isochromen-6-one. It is an isomer of Monascusin E. 10μM Monascusin F reduced the transcription levels of IL-1β, TNF-α, and TGF-β in LPS-induced RAW264.7 cells by 77%, 86%, and 73%, respectively, which is stronger than the positive control DEX with anti-inflammatory immune activity at the same concentration. The structural formula of Monascusin F is as follows:

[0129]

[0130] Example 2

[0131] The steps for extracting monascus D, E, and F from Monascus RCEF7056 are as follows:

[0132] (1) Strain domestication

[0133] The Monascus spores were dissolved in 0.5% Tween 80 and then diluted with sterile water to a concentration of 10 7The spore suspension of spores / mL was inoculated into 250mL of PDB medium at 3% per bottle, and cultured in a shake flask at 35°C and 250r / min for 10 days. Then, the culture was inoculated into a freshly prepared 250mL of PDB medium at 3% and cultured in a shake flask at 35°C and 250r / min for 10 days. Then, the culture was inoculated into a freshly prepared 250mL of PDB medium at 3% and cultured in a shake flask at 35°C and 250r / min for 10 days. Then, the previous step was repeated, and the culture was inoculated into a freshly prepared 250mL of PDB medium at 3% and cultured in a shake flask at 35°C and 250r / min for 10 days. This inoculation culture was repeated 9 times in total. Finally, the culture was inoculated onto potato agar (PDA) medium, and 1000 μL of the culture was inoculated into each culture dish (11 cm in diameter). The culture was cultured at 35° C. for 10 days, and the spores were collected to obtain the domesticated red yeast rice strain RCEF7056.

[0134] (2) Preparation of solid culture

[0135] The liquid strain of Monascus RCEF7056 is subjected to primary strain culture, secondary seed culture and tertiary liquid expansion culture to obtain a liquid culture;

[0136] (2.1) Primary bacterial culture

[0137] The spore suspension of Monascus anka was inoculated on a potato agar (PDA) slant culture medium, with each slant inoculated with 30 μL of the spore suspension. The culture was carried out at a temperature of 35° C. and a rotation speed of 250 r / min for 9 days to obtain a primary strain.

[0138] PDA medium is potato dextrose medium, and its formula is: 200g peeled potato, 20g glucose and 15g agar, add water to 1000mL.

[0139] (2.2) Secondary seed culture

[0140] Dissolve the primary strain in 0.5% Tween 80 and then make it into 10% with sterile water. 7 The spore suspension with a concentration of spores / mL was inoculated into 250 mL of PDB medium at a rate of 250 μL per bottle, and the culture was carried out in a shaking flask at a temperature of 35° C. and a rotation speed of 250 r / min for 5 days to obtain a liquid secondary strain.

[0141] The formula of PDB medium is: 200 g peeled potato, 20 g glucose and water to 1000 mL.

[0142] (2.3) Tertiary liquid expansion culture

[0143] 3 mL of the liquid secondary bacterial strain was inoculated into 250 mL of PDB medium, and cultured in a shaking flask at a temperature of 35° C. and a rotation speed of 250 r / min for 9 days. The culture was collected to obtain a liquid culture.

[0144] (3) Extraction and purification of active ingredients from liquid cultures

[0145] The specific operations are as follows:

[0146] (3.1) Extraction of active ingredients from liquid culture

[0147] The culture was concentrated under reduced pressure and freeze-dried, and then extracted with methanol to obtain an effective component extract.

[0148] The specific operation is as follows: the liquid culture is evaporated under reduced pressure to 1 / 10 of the original volume at a vacuum degree of -0.08MP and a temperature of 60°C, dried at a temperature of 130°C, and crushed; methanol is added at a weight volume ratio of 1g:5mL, and ultrasonic extraction is performed at 40KHz for 200 minutes. Then centrifugation is performed at a speed of 15,000 rpm, and the supernatant is evaporated under reduced pressure at a vacuum degree of -0.08MP and a temperature of 60°C to obtain an effective component extract.

[0149] (3.2) Preliminary purification of active ingredient extracts

[0150] The extract was preliminarily purified by preparative reverse-phase high performance liquid chromatography to obtain a red preliminarily purified product and a yellow preliminarily purified product.

[0151] The specific operation is as follows: purification is carried out by preparative reverse-phase high performance liquid chromatography; the chromatographic separation preparation conditions are: mobile phase A is ultrapure water, and mobile phase B is acetonitrile; elution conditions: 40% to 70% mobile phase B gradient elution from 0 to 30min; injection volume is 300μL, column temperature is 40°C, flow rate is 50mL / min; detection wavelength is 358nm; chromatographic column: preparative reverse-phase C-18 column; collect the red eluate and the yellow eluate, evaporate the extractant under reduced pressure at a vacuum degree of -0.08MP and a temperature of 60°C, and dry at 130°C to obtain a red preliminary purified product and a yellow preliminary purified product.

[0152] (3.3) Purification of the Preliminary Purification Product

[0153] The two preliminary purified products were refined by semi-preparative reverse-phase high performance liquid chromatography. Under the elution condition of acetonitrile solution, the compounds corresponding to the highest peaks were collected, which were the eluents of Monascus D, Monascus E and F. After evaporating the solvent, red Monascus D and yellow Monascus E and F were obtained.

[0154] The chromatographic separation preparation conditions are as follows: chromatographic mobile phase A is ultrapure water, and mobile phase B is acetonitrile; elution conditions: elution with 40% to 70% mobile phase B from 0 to 30 min; injection volume is 200 μL, column temperature is 40°C, flow rate is 20 mL / min; detection wavelength is 358 nm; chromatographic column: semi-preparative reverse phase C-18 column.

[0155] The specific operations are as follows:

[0156] Inject 200 mg / mL of the red preliminary purified product, collect the eluate corresponding to the highest chromatographic peak, and obtain the red chromatographic dye D eluate; remove the extractant by reduced pressure evaporation of the eluate under vacuum degree of -0.08 MP and temperature of 60° C., and dry at 130° C. to obtain red solid red chromatographic dye D.

[0157] 200 mg / mL of the red preliminary purified product was injected, and the eluents corresponding to the first highest chromatographic peak and the second highest chromatographic peak were collected in turn to obtain the red chromatographic eluate and the red chromatographic eluate, respectively; the red chromatographic eluate and the red chromatographic eluate were evaporated under reduced pressure at a vacuum degree of -0.08 MP and a temperature of 60°C to remove the extractant, and dried at 130°C; yellow solid red chromatographic E and yellow solid red chromatographic F were obtained, respectively.

[0158] Monascus succinate D is a red powder with a molecular formula of C 25 H 31 NO 5 . 10μM red yeast rice D reduced the transcription levels of IL-1β, TNF-α, and TGF-β in LPS-induced RAW264.7 cells by 81%, 75%, and 68%, respectively, which was stronger than the positive control DEX with the same concentration of anti-inflammatory immune activity. The structural formula of red yeast rice D is as follows:

[0159]

[0160] Monascus E is a yellow powder with a molecular formula of C 22 H 32 O 5 , chemical name is (7R,8R)-7-hydroxy-3-(2-hydroxypropy)-7-methyl-8-(2-oxonony)-7,8-dihydro-6H-isochromen-6-one. 10μM red yeast rice E reduced the transcription levels of IL-1β, TNF-α, and TGF-β in LPS-induced RAW264.7 cells by 67%, 59%, and 61%, respectively, which is close to the activity of the positive control DEX of the same concentration of anti-inflammatory immune activity. The structural formula of red yeast rice E is as follows:

[0161]

[0162] Monascusin F is a yellow powder with a chemical name of (7S,8S)-7-hydroxy-3-(2-hydroxypropy)-7-methyl-8-(2-oxonony)-7,8-dihydro-6H-isochromen-6-one. It is an isomer of Monascusin E. 10μM Monascusin F reduced the transcription levels of IL-1β, TNF-α, and TGF-β in LPS-induced RAW264.7 cells by 77%, 86%, and 73%, respectively, which is stronger than the positive control DEX with anti-inflammatory immune activity at the same concentration. The structural formula of Monascusin F is as follows:

[0163]

[0164] Example 3

[0165] The steps for extracting monascus D, E, and F from Monascus RCEF7056 are as follows:

[0166] (1) Strain domestication

[0167] The Monascus spores were dissolved in 0.5% Tween 80 and then diluted with sterile water to a concentration of 10 6 The spore suspension of spores / mL was inoculated into 250mL of PDB medium at 2% per bottle, and cultured in a shake flask at 33°C and 230r / min for 8 days. Then, the culture was inoculated into a freshly prepared 230mL of PDB medium at 2% and cultured in a shake flask at 33°C and 230r / min for 8 days. Then, the culture was inoculated into a freshly prepared 250mL of PDB medium at 2% and cultured in a shake flask at 33°C and 230r / min for 8 days. Then, the previous step was repeated, and the culture was inoculated into a freshly prepared 250mL of PDB medium at 2% and cultured in a shake flask at 33°C and 230r / min for 8 days. The inoculation culture was repeated 7 times. Finally, the culture was inoculated onto potato agar (PDA) medium, and 600 μL of the culture was inoculated into each culture dish (11 cm in diameter). The culture was cultured at 33° C. for 8 days, and the spores were collected to obtain the domesticated red yeast rice strain RCEF7056.

[0168] (2) Preparation of solid culture

[0169] The strain of Monascus RCEF7056 is subjected to primary strain culture, secondary seed culture and tertiary liquid expansion culture to obtain a liquid culture;

[0170] (2.1) Primary bacterial culture

[0171] The spore suspension of Monascus rubrum was inoculated on potato agar (PDA) slant medium, with 20 μL of spore suspension inoculated on each slant, and cultured for 8 days at a temperature of 33.5°C and a rotation speed of 235 r / min to obtain the primary strain.

[0172] PDA medium is potato dextrose medium, and its formula is: 200g peeled potato, 20g glucose and 15g agar, add water to 1000mL.

[0173] (2.2) Secondary seed culture

[0174] Dissolve the primary strain in 0.5% Tween 80 and then make it into 10% with sterile water. 6 The spore suspension with a concentration of spores / mL was inoculated into 250 mL of PDB medium at a rate of 225 μL per bottle, and cultured in a shaking flask at a temperature of 33.5°C and a rotation speed of 240 r / min for 4 days to obtain a liquid secondary strain.

[0175] The formula of PDB medium is: 200g peeled potatoes, 20g glucose, add water to 1000mL.

[0176] (2.3) Tertiary liquid expansion culture

[0177] 2 mL of the liquid secondary bacterial strain was inoculated into 250 mL of PDB medium, and cultured in a shaking flask at a temperature of 33.5° C. and a rotation speed of 240 r / min for 8 days. The culture was collected to obtain a liquid culture.

[0178] (3) Extraction and purification of active ingredients from liquid cultures

[0179] The specific operations are as follows:

[0180] (3.1) Extraction of active ingredients from liquid culture

[0181] The liquid culture was concentrated under reduced pressure and freeze-dried, and extracted with an extractant, wherein the extractant was methanol:ethyl acetate=1:1 (v / v), to obtain an effective component extract.

[0182] The specific operation is as follows: the liquid culture is evaporated to 2 / 10 of the original volume under vacuum degree -0.09MP and temperature 40℃, dried at temperature 70℃, and crushed; the extractant is added at a weight volume ratio of 1g:2.5mL, and ultrasonic extraction is performed at 40KHz for 100 minutes. Then centrifugation is performed at 10,000 rpm, and the extractant is evaporated under vacuum degree -0.09MP and temperature 40℃ to obtain the effective component extract.

[0183] (3.2) Preliminary purification of active ingredient extracts

[0184] The active ingredient extract was preliminarily purified by preparative reverse-phase high performance liquid chromatography to obtain a red preliminarily purified product and a yellow preliminarily purified product.

[0185] The specific operation is: using preparative reverse-phase high performance liquid chromatography for purification; the chromatographic separation preparation conditions are: mobile phase A is ultrapure water, and mobile phase B is acetonitrile; elution conditions: 0 to 30min 40% to 70% mobile phase B gradient elution; injection volume 180μL, column temperature 30°C, flow rate 25mL / min; detection wavelength is 358nm; chromatographic column: preparative reverse-phase C-18 column; collecting the red eluent and the yellow eluent, under vacuum degree -0.09MP, temperature 40°C, the extractant is evaporated under reduced pressure, and dried at 70°C to obtain a red preliminary purified product and a yellow preliminary purified product.

[0186] (3.3) Purification of the Preliminary Purification Product

[0187] The two preliminary purified products were refined by semi-preparative reverse-phase high performance liquid chromatography. Under the elution condition of acetonitrile solution, the compounds corresponding to the chromatographic peaks were collected, which were the eluent of Monascus D, Monascus E and F. After evaporating and removing the solvent, red Monascus D and yellow Monascus E and F were obtained.

[0188] The chromatographic separation preparation conditions are as follows: chromatographic mobile phase A is ultrapure water, and mobile phase B is acetonitrile; elution conditions: elution with 40% to 70% mobile phase B from 0 to 30 min; injection volume is 100 μL, column temperature is 30°C, flow rate is 10 mL / min; detection wavelength is 358 nm; chromatographic column: semi-preparative reverse phase C-18 column.

[0189] The specific operations are as follows:

[0190] 100 mg / mL of the red preliminary purified product was injected, and the eluate corresponding to the highest chromatographic peak was collected to obtain the red chromatographic dye D eluate; the eluate was evaporated under reduced pressure at a vacuum degree of -0.09 MP and a temperature of 30° C. to remove the extractant, and then dried at 60° C. to obtain a red solid red chromatographic dye D.

[0191] 100 mg / mL of the red preliminary purified product was injected, and the eluents corresponding to the first highest chromatographic peak and the second highest chromatographic peak were collected in turn to obtain the eluent of Monascus E and the eluent of Monascus F, respectively; the eluent of Monascus E and the eluent of Monascus F were evaporated under reduced pressure at a vacuum degree of -0.09 MP and a temperature of 30°C to remove the extractant, and dried at 60°C; yellow solid Monascus E and yellow solid Monascus F were obtained, respectively.

[0192] Monascus succinate D is a red powder with a molecular formula of C 25 H 31 NO 510μM red yeast rice D reduced the transcription levels of IL-1β, TNF-α, and TGF-β in LPS-induced RAW264.7 cells by 81%, 75%, and 68%, respectively, which was stronger than the positive control DEX with the same concentration of anti-inflammatory immune activity. The structural formula of red yeast rice D is as follows:

[0193]

[0194] Monascus E is a yellow powder with a molecular formula of C 22 H 32 O 5 , chemical name is (7R,8R)-7-hydroxy-3-(2-hydroxypropy)-7-methyl-8-(2-oxonony)-7,8-dihydro-6H-isochromen-6-one. 10μM red yeast rice E reduced the transcription levels of IL-1β, TNF-α, and TGF-β in LPS-induced RAW264.7 cells by 67%, 59%, and 61%, respectively, which is close to the activity of the positive control DEX of the same concentration of anti-inflammatory immune activity.

[0195] The structural formula of red yeast rice is as follows:

[0196]

[0197] Monascusin F is a yellow powder with a chemical name of (7S,8S)-7-hydroxy-3-(2-hydroxypropy)-7-methyl-8-(2-oxonony)-7,8-dihydro-6H-isochromen-6-one. It is an isomer of Monascusin E. 10μM Monascusin F reduced the transcription levels of IL-1β, TNF-α, and TGF-β in LPS-induced RAW264.7 cells by 77%, 86%, and 73%, respectively, which is stronger than the positive control DEX with anti-inflammatory immune activity at the same concentration. The structural formula of Monascusin F is as follows:

[0198]

[0199] Example 4

[0200] The steps for extracting monascus D, E, and F from Monascus RCEF7056 are as follows:

[0201] (1) Strain domestication

[0202] The Monascus spores were dissolved in 0.5% Tween 80 and then diluted with sterile water to a concentration of 10 6The spore suspension of spores / mL was inoculated into 250mL of PDB medium at 2% per bottle, and cultured in a shake flask at 34°C and 240r / min for 9 days. Then, the culture was inoculated into a freshly prepared 240mL of PDB medium at 2% and cultured in a shake flask at 34°C and 240r / min for 9 days. Then, the culture was inoculated into a freshly prepared 250mL of PDB medium at 2% and cultured in a shake flask at 34°C and 240r / min for 9 days. Then, the previous step was repeated, and the culture was inoculated into a freshly prepared 250mL of PDB medium at 2% and cultured in a shake flask at 34°C and 220-250r / min for 9 days. The inoculation culture was repeated 8 times in total. Finally, the culture was inoculated onto potato agar (PDA) medium, and 800 μL of the culture was inoculated into each culture dish (11 cm in diameter). The culture was cultured at 34° C. for 9 days, and the spores were collected to obtain the domesticated red yeast rice strain RCEF7056.

[0203] (2) Preparation of solid culture

[0204] The strain of Monascus ruber is subjected to primary strain culture, secondary seed culture and tertiary liquid expansion culture to obtain a liquid culture;

[0205] (2.1) Primary bacterial culture

[0206] The spore suspension of Monascus rubrum was inoculated on a potato agar (PDA) slant culture medium, with each slant inoculated with 20 μL of the spore suspension. The culture was carried out at a temperature of 33.5° C. and a rotation speed of 240 r / min for 8 days to obtain a primary strain.

[0207] PDA medium is potato dextrose medium, and its formula is: 200g peeled potato, 20g glucose and 15g agar, add water to 1000mL.

[0208] (2.2) Secondary seed culture

[0209] Dissolve the primary strain in 0.5% Tween 80 and then make it into 10% with sterile water. 6 The spore suspension with a concentration of spores / mL was inoculated into 250 mL of PDB medium at a rate of 220 μL per bottle, and cultured in a shaking flask at a temperature of 33°C and a rotation speed of 240 r / min for 4 days to obtain a liquid secondary strain.

[0210] The formula of PDB medium is: 200g peeled potatoes, 20g glucose, add water to 1000mL.

[0211] (2.3) Tertiary liquid expansion culture

[0212] 2 mL of the liquid secondary bacterial strain was inoculated into 250 mL of PDB medium, and cultured in a shaking flask at a temperature of 33.5° C. and a rotation speed of 235 r / min for 8 days. The culture was collected to obtain a liquid culture.

[0213] (3) Extraction and purification of active ingredients from liquid cultures

[0214] The specific operations are as follows:

[0215] (3.1) Extraction of active ingredients from liquid culture

[0216] The culture was concentrated and dried under reduced pressure, and then homogenized and extracted with methanol:ethyl acetate=0.5:1 (v / v) to obtain an effective component extract.

[0217] The specific operation is: the liquid culture is evaporated under reduced pressure to 3 / 10 of the original volume under the conditions of vacuum degree -0.1MP and temperature 10℃, and then directly extracted with the extractant for 20 minutes. Then, it is filtered with a 0.1μm membrane, and the filtrate is evaporated under reduced pressure under the conditions of vacuum degree -0.1MP and temperature 10℃ to remove the extractant, and the effective component extract is obtained.

[0218] (3.2) Preliminary purification of active ingredient extracts

[0219] The active ingredient extract was preliminarily purified by preparative reverse-phase high performance liquid chromatography to obtain a red preliminarily purified product and a yellow preliminarily purified product.

[0220] The specific operation is as follows: purification is carried out by preparative reverse-phase high performance liquid chromatography; the chromatographic separation preparation conditions are: mobile phase A is ultrapure water, and mobile phase B is acetonitrile; elution conditions: gradient elution of 40% to 70% mobile phase B from 0 to 30 min; injection volume is 200 μL, column temperature is 30°C, flow rate is 20 mL / min; detection wavelength is 358 nm; chromatographic column: preparative reverse-phase C-18 column; red eluent and yellow eluent are collected, and the extractant is evaporated under reduced pressure at a vacuum degree of -0.1 MP and a temperature of 30°C, and dried at -40°C to obtain a red preliminary purified product and a yellow preliminary purified product.

[0221] (3.3) Purification of the Preliminary Purification Product

[0222] The two preliminary purified products were refined by semi-preparative reverse-phase high performance liquid chromatography. Under the elution condition of acetonitrile solution, the compounds corresponding to the chromatographic peaks were collected, which were monascus D, monascus E and F eluates. After evaporating and removing the solvent, red monascus D and yellow monascus E and F were obtained.

[0223] The chromatographic separation preparation conditions are: the chromatographic separation preparation conditions are: mobile phase A is ultrapure water, mobile phase B is acetonitrile; elution conditions: elution with 40% to 70% mobile phase B from 0 to 30 min; injection volume is 30 μL, column temperature is 30°C, flow rate is 5 mL / min; detection wavelength is 358 nm; chromatographic column: semi-preparative reverse phase C-18 column.

[0224] The specific operations are as follows:

[0225] Inject 150 mg / mL of the red preliminary purified product, collect the eluate corresponding to the highest chromatographic peak, and obtain the red chromatographic dye D eluate; remove the extractant by reduced pressure evaporation of the eluate under vacuum degree of -0.1 MP and temperature of 20°C, and dry at 40°C; obtain red solid red chromatographic dye D.

[0226] 150 mg / mL of the red preliminary purified product was injected, and the eluents corresponding to the first highest chromatographic peak and the second highest chromatographic peak were collected in turn to obtain the red chromatographic eluate and the red chromatographic F eluate, respectively; the red chromatographic eluate and the red chromatographic F eluate were respectively evaporated under reduced pressure at a vacuum degree of -0.1 MP and a temperature of 20°C to remove the extractant, and dried at 40°C; yellow solid red chromatographic E and yellow solid red chromatographic F were obtained, respectively.

[0227] Monascus succinate D is a red powder with a molecular formula of C 25 H 31 NO 5 10μM red yeast rice D reduced the transcription levels of IL-1β, TNF-α, and TGF-β in LPS-induced RAW264.7 cells by 81%, 75%, and 68%, respectively, which was stronger than the positive control DEX with the same concentration of anti-inflammatory immune activity. The structural formula of red yeast rice D is as follows:

[0228]

[0229] Monascus E is a yellow powder with a molecular formula of C 22 H 32 O 5 , chemical name is (7R,8R)-7-hydroxy-3-(2-hydroxypropy)-7-methyl-8-(2-oxonony)-7,8-dihydro-6H-isochromen-6-one. 10μM red yeast rice E reduced the transcription levels of IL-1β, TNF-α, and TGF-β in LPS-induced RAW264.7 cells by 67%, 59%, and 61%, respectively, which is close to the activity of the positive control DEX of the same concentration of anti-inflammatory immune activity.

[0230] The structural formula of red yeast rice is as follows:

[0231]

[0232] Monascusin F is a yellow powder with a chemical name of (7S,8S)-7-hydroxy-3-(2-hydroxypropy)-7-methyl-8-(2-oxonony)-7,8-dihydro-6H-isochromen-6-one. It is an isomer of Monascusin E. 10μM Monascusin F reduced the transcription levels of IL-1β, TNF-α, and TGF-β in LPS-induced RAW264.7 cells by 77%, 86%, and 73%, respectively, which is stronger than the positive control DEX with anti-inflammatory immune activity at the same concentration. The structural formula of Monascusin F is as follows:

[0233]

[0234] Example 5

[0235] The steps for extracting monascus D, E, and F from Monascus RCEF7056 are as follows:

[0236] (1) Strain domestication

[0237] The Monascus spores were dissolved in 0.5% Tween 80 and then diluted with sterile water to a concentration of 10 6 The spore suspension of spores / mL was inoculated into 250mL of PDB medium at 2% per bottle, and cultured in a shake flask at 33°C and 230r / min for 9 days. Then, the culture was inoculated into a freshly prepared 230mL of PDB medium at 2% and cultured in a shake flask at 33°C and 230r / min for 9 days. Then, the culture was inoculated into a freshly prepared 250mL of PDB medium at 2% and cultured in a shake flask at 33°C and 230r / min for 9 days. Then, the previous step was repeated, and the culture was inoculated into a freshly prepared 250mL of PDB medium at 2% and cultured in a shake flask at 32°C and 230r / min for 9 days. The inoculation culture was repeated 7 times. Finally, the culture was inoculated onto potato agar (PDA) medium, and each culture dish (11 cm in diameter) was inoculated with 700 μL of the culture. The culture was cultured at 33° C. for 9 days, and the spores were collected to obtain the domesticated red yeast rice original species RCEF7056.

[0238] (2) Preparation of solid culture

[0239] The strain of Monascus ruber is subjected to primary strain culture, secondary seed culture and tertiary liquid expansion culture to obtain a liquid culture;

[0240] (2.1) Primary bacterial culture

[0241] The spore suspension of Monascus rubrum was inoculated on a potato agar (PDA) slant culture medium, with each slant inoculated with 20 μL of the spore suspension. The culture was carried out at a temperature of 33.5° C. and a rotation speed of 240 r / min for 8 days to obtain a primary strain.

[0242] PDA medium is potato dextrose medium, and its formula is: 200g peeled potato, 20g glucose and 15g agar, add water to 1000mL.

[0243] (2.2) Secondary seed culture

[0244] Dissolve the primary strain in 0.5% Tween 80 and then make it into 10% with sterile water. 6 The spore suspension with a concentration of spores / mL was inoculated into 250 mL of PDB medium at a rate of 220 μL per bottle, and cultured in a shaking flask at a temperature of 33°C and a rotation speed of 240 r / min for 4 days to obtain a liquid secondary strain.

[0245] The formula of PDB medium is: 200g peeled potatoes, 20g glucose, add water to 1000mL.

[0246] (2.3) Tertiary liquid expansion culture

[0247] 2 mL of liquid secondary bacteria was inoculated into 250 mL of PDB medium, and cultured in a shaking flask at a temperature of 33.5° C. and a rotation speed of 235 r / min for 8 days. The culture was collected to obtain a liquid culture.

[0248] (3) Extraction and purification of active ingredients from liquid cultures

[0249] The specific operations are as follows:

[0250] (3.1) Extraction of active ingredients from liquid culture

[0251] The culture was concentrated under reduced pressure, homogenized, and extracted with methanol:ethyl acetate=1:0.5 (v / v) to obtain an effective component extract.

[0252] The specific operation is as follows: the liquid culture is evaporated under reduced pressure to 3 / 10 to 1 / 10 of the original volume under the conditions of vacuum degree -0.1 to -0.08 MP and temperature 10 to 60°C, and directly extracted with ethyl acetate for 20 to 200 minutes. Then, it is filtered with a 2.5 μm membrane, and the filtrate is evaporated under reduced pressure to remove the extractant under the conditions of vacuum degree -0.08 MP and temperature 60°C to obtain the effective component extract.

[0253] (3.2) Preliminary purification of active ingredient extracts

[0254] The extract was preliminarily purified by preparative reverse-phase high performance liquid chromatography to obtain a red preliminarily purified product and a yellow preliminarily purified product.

[0255] The specific operation is as follows: purification is carried out by preparative reverse-phase high performance liquid chromatography; the chromatographic separation preparation conditions are: mobile phase A is ultrapure water, and mobile phase B is acetonitrile; elution conditions: gradient elution of 40% to 70% mobile phase B from 0 to 30 min; injection volume is 200 μL, column temperature is 30°C, flow rate is 20 mL / min; detection wavelength is 358 nm; chromatographic column: preparative reverse-phase C-18 column; red eluent and yellow eluent are collected, and the extractant is evaporated under reduced pressure at a vacuum degree of -0.1 MP and a temperature of 30°C, and dried at -40°C to obtain a red preliminary purified product and a yellow preliminary purified product.

[0256] (3.3) Purification of the Preliminary Purification Product

[0257] The two preliminary purified products were refined by semi-preparative reverse-phase high performance liquid chromatography. Under the elution condition of acetonitrile solution, the compounds corresponding to the chromatographic peaks were collected, which were monascus D, monascus E and F eluates. After evaporating and removing the solvent, red monascus D and yellow monascus E and F were obtained.

[0258] The chromatographic separation preparation conditions are: the chromatographic separation preparation conditions are: mobile phase A is ultrapure water, mobile phase B is acetonitrile; elution conditions: elution with 40% to 70% mobile phase B from 0 to 30 min; injection volume is 30 μL, column temperature is 30°C, flow rate is 5 mL / min; detection wavelength is 358 nm; chromatographic column: semi-preparative reverse phase C-18 column.

[0259] The specific operations are as follows:

[0260] 80 mg / mL of the red preliminary purified product was injected, and the eluate corresponding to the highest chromatographic peak was collected to obtain the red chromatographic dye D eluate; the eluate was evaporated under reduced pressure at a vacuum degree of -0.1 MP and a temperature of 40°C to remove the extractant, and dried at 80°C to obtain a red solid red chromatographic dye D.

[0261] 80 mg / mL of the red preliminary purified product was injected, and the eluents corresponding to the first highest chromatographic peak and the second highest chromatographic peak were collected in turn to obtain the eluent of Monascus E and the eluent of Monascus F, respectively; these eluents were evaporated under reduced pressure at a vacuum degree of -0.1 MP and a temperature of 40°C to remove the extractant, and then dried at 80°C; yellow solid Monascus E and yellow solid Monascus F were obtained, respectively.

[0262] Monascus succinate D is a red powder with a molecular formula of C 25 H 31 NO 510μM red yeast rice D reduced the transcription levels of IL-1β, TNF-α, and TGF-β in LPS-induced RAW264.7 cells by 81%, 75%, and 68%, respectively, which was stronger than the positive control DEX with the same concentration of anti-inflammatory immune activity. The structural formula of red yeast rice D is as follows:

[0263]

[0264] Monascus E is a yellow powder with a molecular formula of C 22 H 32 O 5 , chemical name is (7R,8R)-7-hydroxy-3-(2-hydroxypropy)-7-methyl-8-(2-oxonony)-7,8-dihydro-6H-isochromen-6-one. 10μM red yeast rice E reduced the transcription levels of IL-1β, TNF-α, and TGF-β in LPS-induced RAW264.7 cells by 67%, 59%, and 61%, respectively, which is close to the activity of the positive control DEX of the same concentration of anti-inflammatory immune activity.

[0265] The structural formula of red yeast rice is as follows:

[0266]

[0267] Monascusin F is a yellow powder with a chemical name of (7S,8S)-7-hydroxy-3-(2-hydroxypropy)-7-methyl-8-(2-oxonony)-7,8-dihydro-6H-isochromen-6-one. It is an isomer of Monascusin E. 10μM Monascusin F reduced the transcription levels of IL-1β, TNF-α, and TGF-β in LPS-induced RAW264.7 cells by 77%, 86%, and 73%, respectively, which is stronger than the positive control DEX with anti-inflammatory immune activity at the same concentration. The structural formula of Monascusin F is as follows:

[0268]

[0269] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A red yeast rice with anti-inflammatory immune activity, characterized in that: The monascusins ​​include monascus D, monascus E and monascus F which are separated and prepared from the liquid culture of Monascus purpureus; The monascusin D is a red powder with a molecular formula of C 25 H 31 NO5; Monascus D has a rare N-ethanol group in its structure. The chemical name of Monascus D is E-7-(2-hydroxyethyl)-9a-methyl-3-octanoyl-6-(prop-1-en-1-yl)furo[3,2- g]isoquinoline-2,9(7H,9aH)-dione, the structural formula of red yeast rice D is as follows: The monascusin E is a yellow powder with a molecular formula of C 22 H 32 O5, chemical name is (7R,8R)-7-hydroxy-3-(2-hydroxypropy)-7-methyl-8-(2-oxonony)-7,8-dihydro-6H-isochromen-6-one; the structural formula of red yeast rice is as follows: The monascusin F is a yellow powder and is an isomer of monascusin E. Its chemical name is (7S,8S)-7-hydroxy-3-(2-hydroxypropy)-7-methyl-8-(2-oxonony)-7,8-dihydro-6H-isochromen-6-one. The structural formula of monascusin F is as follows: The Monascus is Monascus purpureus; the Monascus RCEF7056 strain is obtained by liquid culture and domestication of Monascus purpureus, the liquid culture is obtained by expanding the culture of the Monascus RCEF7056 strain, and the liquid culture is separated and purified to obtain Monascus D, Monascus E and Monascus F; 10 μM red yeast rice D reduced the transcription levels of IL-1β, TNF-α, and TGF-β in LPS-induced RAW264.7 cells by 81%, 75%, and 68%, respectively, which was stronger than the positive control dexamethasone (DEX) with the same concentration of anti-inflammatory immune activity; 10 μM red yeast rice E reduced the transcription levels of IL-1β, TNF-α, and TGF-β in LPS-induced RAW264.7 cells by 67%, 59%, and 61%, respectively, which was close to the activity of the positive control dexamethasone (DEX) with anti-inflammatory immune activity at the same concentration; 10 μM red yeast rice F reduced the transcription levels of IL-1β, TNF-α, and TGF-β in LPS-induced RAW264.7 cells by 77%, 86%, and 73%, respectively, which was stronger than the positive control dexamethasone (DEX) with anti-inflammatory immune activity at the same concentration.

2. A method for preparing red yeast rice with anti-inflammatory immune activity as claimed in claim 1, characterized in that: The steps are as follows: (1) Bacteria domestication (1.1) Dissolve the Monascus spores in 0.5% Tween 80 and then add sterile water to a concentration of 10 5～7 spores / mL of spore suspension, inoculated into 250mL of PDB liquid culture medium at 1-3% of the volume of each bottle, and cultured in a shaking bottle at a temperature of 32-35°C and a rotation speed of 220-250r / min for 7-10 days; repeating the cycle of inoculation and culture 5-9 times to obtain an acclimated liquid culture; (1.2) inoculating the domesticated liquid culture onto a potato agar (PDA) medium, inoculating 200-1000 μL of the domesticated liquid culture into each culture dish of the potato agar (PDA) medium (11 cm in diameter), culturing at a temperature of 32-35° C. for 7-10 days, collecting spores, and obtaining liquid culture domesticated Monascus RCEF7056 strains; (2) Preparation and expansion of liquid culture The spore suspension of Monascus RCEF7056 is subjected to primary strain culture, secondary seed culture and tertiary liquid expansion culture to obtain an expanded liquid culture; The primary bacterial culture is potato agar (PDA) slant culture; The secondary seed culture is a liquid culture of PDB medium; The tertiary liquid expansion culture is a liquid culture of PDB medium; (3) Expanding the extraction and purification of active ingredients from liquid cultures The extractant used for the extraction and purification is prepared by uniformly mixing methanol and ethyl acetate in a volume ratio of 0-1:1-0; the specific extraction and purification operations are as follows: (3.1) Extraction of active ingredients from liquid culture The expanded liquid culture is concentrated and dried under reduced pressure; ultrasonic extraction is performed using an extractant, membrane filtration or centrifugal separation is performed, and the solvent is removed by reduced pressure evaporation to obtain an effective component extract; (3.2) Preliminary purification of active ingredient extract Preliminary purification of the active ingredient extract was performed using preparative reverse-phase high performance liquid chromatography to obtain a red preliminary purified product and a yellow preliminary purified product; (3.3) Purification of two preliminary purified products The two preliminary purified products were purified separately by semi-preparative reversed-phase HPLC; The red preliminary purified product was eluted with acetonitrile solution, and the compound corresponding to the highest chromatographic peak was collected as the monascus D eluate, and the solvent was evaporated to obtain red monascus D; The yellow preliminary purified product was eluted with acetonitrile solution, and the eluates corresponding to the first highest chromatographic peak and the second highest chromatographic peak were collected, namely, the eluate of Monascus E and the eluate of Monascus F, respectively. The solvent was evaporated to obtain yellow Monascus E and yellow Monascus F.

3. The preparation method according to claim 2, characterized in that: In step (2), the specific operations are as follows: (2.1) Primary bacterial culture, The spore suspension of Monascus RCEF7056 strain is 10 6 The preparation method of the Monascus spore suspension is as follows: 0.1 g of spores are dissolved in 10 mL of 0.5% Tween 80, and then the spore concentration is diluted to 10 with sterile water. 6 spores / mL; The spore suspension of Monascus RCEF7056 strain is inoculated on a potato agar (PDA) slant medium, each slant is inoculated with 10-30 μL of the spore suspension, and cultured at a temperature of 32-35° C. and 220-250 r / min for 7-9 days, and the spores are collected to obtain a primary strain; (2.2) Secondary seed culture Dissolve the primary strain with 0.5% Tween 80 and dilute to 10% with sterile water. 5 ~10 7 spores / mL of spore suspension, inoculated into 250mL of liquid PDB medium at an inoculum volume of 200-250μL per bottle, and cultured in a shaking flask at a temperature of 32-35°C and a rotation speed of 220-250r / min for 3-5 days to obtain a secondary liquid strain; (2.3) Tertiary liquid expansion culture 1-3 mL of the secondary liquid bacterial strain was inoculated into 250 mL of liquid PDB medium, and cultured in a shaking flask at a temperature of 32-35° C. and a rotation speed of 220-250 r / min for 7-9 days to obtain a liquid culture.

4. The preparation method according to claim 2, characterized in that: In step (3.1), the specific operation is as follows: the liquid culture is concentrated under reduced pressure to 3 / 10 to 1 / 10 of the original volume under vacuum conditions of -0.1 to -0.08 MP and temperature of 10 to 60°C; dried and crushed under temperature conditions of -50 to 130°C, an extractant is added at a weight volume ratio of 1 g: 0.5 to 5 mL, and ultrasonic extraction is performed at 40KHz for 20 to 200 minutes; centrifuged at 4000 to 15000 rpm, or filtered through a 0.1 to 2.5 μm membrane to obtain a filtrate or a centrifugal supernatant; the filtrate or the centrifugal supernatant is evaporated under reduced pressure to remove the extractant under vacuum conditions of -0.1 to -0.08 MP and temperature of 10 to 60°C to obtain an effective ingredient extract.

5. The preparation method according to claim 2, characterized in that: In step (3.2), the specific operations are as follows: The method adopts preparative reverse phase high performance liquid chromatography for purification; the chromatographic separation preparation conditions are as follows: mobile phase A is ultrapure water, and mobile phase B is acetonitrile; the elution conditions are as follows: 40% to 70% mobile phase B gradient elution from 0 to 30 minutes; the injection volume is 30 to 300 μL, the column temperature is 20 to 40° C., and the flow rate is 5 to 50 mL / min; the detection wavelength is 358 nm; the chromatographic column is a preparative reverse phase C-18 column; the red eluent and the yellow eluent are collected, and the extractant is evaporated under reduced pressure under the conditions of a vacuum degree of -0.1 to -0.08 MP and a temperature of 10 to 60° C., and the product is dried at a temperature of less than or equal to 130° C. to obtain a red preliminary purified product and a yellow preliminary purified product.

6. The preparation method according to claim 2, characterized in that: In step (3.3), the specific operations are as follows: The chromatographic separation preparation conditions are as follows: mobile phase A is ultrapure water, mobile phase B is acetonitrile; elution conditions: elution with 40% to 70% mobile phase B from 0 to 30 min; injection volume 20 to 200 μL, column temperature 20 to 40° C., flow rate 2 to 20 mL / min; detection wavelength 358 nm; Chromatographic column: semi-preparative reverse phase C-18 column; Inject 20-200 mg / mL of the red preliminary purified product, collect the eluate corresponding to the highest chromatographic peak, and obtain the red chromatographic dye D eluate; remove the extractant by decompression evaporation of the eluate under the conditions of vacuum degree -0.1-0.08 MP and temperature 10-60° C., and dry at 130° C. or less; obtain the red solid red chromatographic dye D; 20-200 mg / mL of the red preliminary purified product is injected, and the eluents corresponding to the first highest chromatographic peak and the second highest chromatographic peak are collected in sequence to obtain the red chromatographic eluent and the red chromatographic eluent respectively; the red chromatographic eluent and the red chromatographic eluent are respectively evaporated under reduced pressure at a vacuum degree of -0.1 to -0.08 MP and a temperature of 10 to 60° C. to remove the extractant, and dried at a temperature of less than or equal to 130° C.; yellow solid red chromatographic E and yellow solid red chromatographic F are respectively obtained.

7. The preparation method according to claim 4, characterized in that: In step (3.1), the extractant is methanol, or ethyl acetate, or a mixture of methanol and ethyl acetate.

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