Monascusin with anti-inflammatory immune activity and preparation method thereof

Abstract

The present invention relates to a monascus with anti-inflammatory and immune activity and a preparation method thereof, belonging to the technical field of extraction and preparation of biological products. The monascus includes monascus D, monascus E, and monascus F, which are separated and prepared from a liquid culture of Monascus purpureus. The Monascus purpureus is obtained by liquid culture and domestication of Monascus purpureus to obtain the Monascus RCEF7056 strain, which is then expanded and cultured to obtain a liquid culture, and monascus D, E, and F are obtained by separation and purification of the liquid culture. Preliminary activity analysis revealed that the monascus 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. The Monascus purpureus is an edible fungus and is safe and reliable. The preparation method of monascus D, E, and F adopts microbial fermentation, is environmentally friendly, is not affected by the natural environment and resources, and is easy to achieve industrialized, automated, and continuous production.

Description

Technical Field

[0001] The present 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 D), Monascuskaolin E (Monascuskaolin E) and Monascuskaolin F (Monascuskaolin F) in a fermentation product of Monascus purpureus. Background Art

[0002] Monascus sp. is a small, filamentous saprophytic fungus belonging to the Eumycophyta, Ascomycotina, Plectomycetes, Eurotiales, Monascaceae, and the genus Monascus. It includes species such as Monascus purpureus, Monascus fuliginosus, and Monascus ruber. Monascus produces a variety of enzymes, including saccharifying enzymes, esterifying enzymes, and proteases. These enzymes work together to produce flavor compounds such as monosaccharides, amino acids, and nucleotides, as well as aroma compounds such as volatile acids, alcohols, and esters, contributing significantly 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. This has seriously affected the safety of foods involving red yeast rice liquid fermentation, such as liquor, rice wine, cooking wine, fermented bean curd and sauce, and also did 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 through long-term liquid fermentation. Preliminary analysis has revealed a large number of novel bioactive substances in this red yeast liquid culture that inhibit inflammation and immunity. In today's rapidly developing society, characterized by a fast-paced and stressful work and life, coupled with a lack of exercise and unhealthy habits such as staying up late to watch their phones, the number of people suffering from immune disorders and chronic inflammation is increasing year by year. Chronic inflammation, in particular, can significantly reduce a person's sense of well-being and can even lead to cancer. Therefore, the development of anti-inflammatory and immune-related drugs is necessary. Monascus, as a food microorganism, offers advantages of safety and reliability, making related immune preparations highly promising and economically valuable. Summary of the Invention

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

[0006] The present invention extracts monascus D, E and F from Monascus RCEF7056 domesticated through liquid culture of red yeast rice. Monascus RCEF7056 is stored in the RCEF bacterial bank, i.e., the World Data Centre for Microorganisms, WDCM No. 1031 bacterial bank. Strain number is 7056.

[0007] A red purpurin with anti-inflammatory immune activity, including red purpurin D, red purpurin E and red purpurin F, is isolated and prepared from a liquid culture of Monascus purpureus;

[0008] The monascusin D is a red powder with a molecular formula of C 25 H 31 NO5; Monascusin D has a rare N-ethanol group in its structure. The chemical name of Monascusin 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 yeast rice D is as follows:

[0010]

[0011] 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 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 the Monascus purpureus, the liquid culture is obtained by expanding the culture of the Monascus RCEF7056 strain, and monascusin D, monascusin E and monascusin F are obtained by separation and purification of the liquid culture;

[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 anti-inflammatory immune activity at the same concentration;

[0017] 10 μM red yeast rice E reduced the transcriptional 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 acclimatization (1.1) Dissolve the Monascus spores with 0.5% Tween 80 and then add sterile water to a concentration of 10 5～7 The spore suspension containing 10 spores / mL is inoculated into 250 mL of PDB liquid culture medium at a rate of 1-3% of the volume of each bottle, and cultured in a shake flask at a temperature of 32-35°C and a rotation speed of 220-250 r / min for 7-10 days; the inoculation and culture cycle is repeated 5-9 times to obtain an acclimated liquid culture;

[0021] (1.2) The acclimated liquid culture was inoculated onto potato agar (PDA) medium. 200-1000 μL of the acclimated liquid culture was inoculated into each PDA dish (11 cm in diameter) and cultured at 32-35°C for 7-10 days. Spores were collected to obtain liquid culture acclimated Monascus RCEF7056 strains.

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

[0023] The spore suspension of Monascus RCEF7056 was subjected to a first-level strain culture, a second-level seed culture and a third-level 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) Expand the extraction and purification of active ingredients from liquid cultures

[0028] The extraction agent used for the extraction and purification is prepared by uniformly mixing methanol and ethyl acetate at 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 under reduced pressure and dried; ultrasonic extraction is performed using an extractant, membrane filtration or centrifugal separation is performed, and the solvent is removed by evaporation under reduced pressure to obtain an active ingredient extract;

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

[0032] 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;

[0033] (3.3) Purify the two preliminary purified products separately

[0034] The two preliminary purified products were purified separately by semi-preparative reversed-phase high performance liquid chromatography;

[0035] The red preliminary purified product was eluted with acetonitrile solution, and the compound corresponding to the highest chromatographic peak was collected as the eluate of Monascus D. 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] 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 the 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 10 to 30 μL of the spore suspension inoculated on each slant. The culture was carried out at a temperature of 32 to 35° C. and a speed of 220 to 250 rpm for 7 to 9 days, and the 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 with sterile water to 10 5 ~10 7 The spore suspension containing spores / mL was inoculated into 250 mL of liquid PDB medium at an inoculum volume of 200-250 μL per bottle. The culture was carried out in a shake flask at a temperature of 32-35°C and a rotation speed of 220-250 r / 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 shake 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 its original volume at a vacuum degree of -0.1 to -0.08 MP and a temperature of 10 to 60°C; dried and crushed at a temperature of -50 to 130°C, an extractant is added at a weight-to-volume ratio of 1 g: 0.5 to 5 mL, and ultrasonic extraction is performed at 40 kHz for 20 to 200 minutes; the liquid culture is 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 extractant is evaporated under reduced pressure from the filtrate or the centrifugal supernatant at 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] Preparative reversed-phase high performance liquid chromatography is used for purification; the chromatographic separation preparation conditions are: mobile phase A is ultrapure water, and mobile phase B is acetonitrile; the elution conditions are: gradient elution of 40% to 70% mobile phase B 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 reversed-phase C-18 column; the red eluate and the yellow eluate are collected, and the extractant is evaporated under reduced pressure under vacuum conditions 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] 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 minutes; injection volume is 20 to 200 μL, column temperature is 20 to 40°C, flow rate is 2 to 20 mL / min; detection wavelength is 358 nm; chromatographic column is a semi-preparative reversed-phase C-18 column;

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

[0053] A 20-200 mg / mL 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 red chromatographic eluents and red chromatographic F eluents, respectively; these eluents are respectively subjected to reduced pressure evaporation to remove the extractant under vacuum conditions 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 red chromatographic E and yellow solid red chromatographic 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 extract. 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 yeast rice was 426.1955, [M+H] + The corresponding molecular formula is C 25 H 31 NO5, 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 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 anti-inflammatory immune activity at the same concentration.

[0064] 4. Chemical structure identification data of Monascusin 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 O5, specific NMR data are shown in Table 2 below.

[0066] Table 2. NMR carbon-hydrogen assignments of red yeast rice E

[0067]

[0068]

[0069] 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 is 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 performed circular dichroism spectroscopy (Circular Dichroism Spectropolarimeter). The results showed that the configuration of red yeast rice 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 yeast rice is as follows:

[0073]

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

[0075] High-resolution liquid chromatography-mass spectrometry analysis showed that the mass-to-charge ratio of the ion of red yeast rice F was 377.2367, [M+H] + The corresponding molecular formula is C 22 H 32 O5, 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 F

[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 , and found that the configuration of red crocus F is (7S, 8S) by circular dichroism spectroscopy. Therefore, 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] In recent years, researchers at home and abroad have discovered numerous bioactive compounds from Monascus purpurogenum (Red Yeast Rice), exhibiting lipid-lowering, antioxidant, anticancer, and antibacterial properties. Some of these compounds have been developed as pharmaceuticals, demonstrating the significant potential of this fungus. Bioinformatics predictions indicate that the genomic DNA of Monascus purpurogenum contains previously unknown gene clusters encoding polyketides and nonribosomal peptides, potentially laying the foundation for the discovery of new secondary metabolites from the fungus. To identify previously unreported compounds in the metabolites of Monascus purpurogenum, the inventors first cultivated a large number of Monascus strains in liquid culture. The active components of the cultures were then extracted and analyzed by liquid chromatography-mass spectrometry (LC-MS). This led to the identification of several new compounds in the metabolites of Monascus RCEF7056. Based on these large-scale cultivation and extraction steps, reverse preparative and semi-preparative chromatography was used to separate and purify Monascus kaolin D, Monascus kaolin E, and Monascus kaolin F. The structural formulas of Monascus D, E, and F were determined by one- and two-dimensional nuclear magnetic resonance (NMR) 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 discovered 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 control, 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 chromatin F. DETAILED DESCRIPTION

[0091] The present invention will be further described below with reference to the 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 A spore suspension containing 10 spores / mL is inoculated into 250 mL of PDB liquid medium at a rate of 1% per bottle, and cultured in a shake flask at a temperature of 32° C. and a rotation speed of 220 r / min for 7 days; the first culture is inoculated into a freshly prepared 220 mL of PDB medium at a rate of 1-3% by volume, and cultured in a shake flask at a temperature of 32° C. and a rotation speed of 220 r / min for 7 days; the second culture is inoculated into a freshly prepared 250 mL of PDB medium at a rate of 1% by volume, and cultured in a shake flask at a temperature of 32° C. and a rotation speed of 220 r / min for 7 days. The above step is then repeated, and the third culture is inoculated into a freshly prepared 250 mL of PDB medium at a rate of 1% by volume, and cultured in a shake flask at a temperature of 32° C. and a rotation speed of 220 r / min for 7 days. This inoculation culture is repeated 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 culture in 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 was 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. 6 spores / mL.

[0102] The spore suspension of Monascus RCEF7056 was inoculated on potato agar (PDA) slant culture medium, with 10 μL of spore suspension inoculated on each slant, and cultured at a temperature of 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 with 0.5% Tween 80 and then add sterile water to a concentration of 10 5 The spore suspension with a concentration of spores / mL was inoculated into 250 mL of liquid PDB medium at a volume of 200 μL per bottle, and cultured 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 potatoes, 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 shake flask at a temperature of 32° C. and a rotation speed of 220 r / min for 7 days. The culture was collected to obtain a liquid culture.

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

[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 procedure is as follows: the liquid culture is evaporated under reduced pressure to 3 / 10 of its original volume at a vacuum of -0.1 MPa and a temperature of 10°C, dried at -50°C, and pulverized. Ethyl acetate is added at a weight-to-volume ratio of 1 g:0.5 mL, and ultrasonic extraction is performed at 40 kHz for 20 minutes. The extract is then centrifuged at 4000 rpm, and the supernatant is evaporated under reduced pressure at a vacuum of -0.1 MPa and a temperature of 10°C to obtain the active ingredient extract.

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

[0115] 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.

[0116] The specific operation is: purification 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: 0 to 30 min, 40% to 70% mobile phase B gradient elution; injection volume 30 μL, column temperature 20°C, flow rate 5 mL / min; detection wavelength is 358 nm; 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 -0.1 MP and a temperature of 10°C, and dry 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, namely, the eluent of Monascus D, Monascus E and F. After evaporation and removal of the solvent, red Monascus D and yellow Monascus E and F were obtained.

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

[0120] The specific operations are as follows:

[0121] 20 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 to remove the extractant under vacuum conditions of -0.1 MP and 10° C., and dried at -45° C. to 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 sequence 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 -0.1 MP and a temperature of 10°C to remove the extractant, and then dried at -45°C to obtain yellow solid red chromatographic E and yellow solid red chromatographic F, respectively.

[0123] Monascusin D is a red powder with the molecular formula C 25 H 31 NO5. 10μM red yeast rice D reduced the transcriptional 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. The structural formula of red yeast rice D is as follows:

[0124]

[0125] Monascusin E is a yellow powder with the molecular formula C 22 H 32 O5, chemically named (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 LPS-induced IL-1β, TNF-α, and TGF-β transcriptional levels in RAW264.7 cells by 67%, 59%, and 61%, respectively. This activity is similar to that of the positive control DEX, which exhibits anti-inflammatory immune activity at the same concentration.

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

[0127]

[0128] Monascusin F is a yellow powder with the chemical name (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 transcriptional levels of IL-1β, TNF-α, and TGF-β in LPS-induced RAW264.7 cells by 77%, 86%, and 73%, respectively. This is more potent than the anti-inflammatory immune control DEX 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 7A spore suspension containing 3 spores / mL was inoculated into 250 mL of PDB medium at a rate of 3% per bottle and cultured in a shake flask at 35°C and 250 r / min for 10 days. Then, 3% of this culture was inoculated into a freshly prepared 250 mL of PDB medium and cultured in a shake flask at 35°C and 250 r / min for 10 days. Then, 3% of this culture was inoculated into a freshly prepared 250 mL of PDB medium and cultured in a shake flask at 35°C and 250 r / min for 10 days. The previous step was then repeated, with 3% of the culture inoculated into a freshly prepared 250 mL of PDB medium and cultured in a shake flask at 35°C and 250 r / min for 10 days. This inoculation culture was repeated a total of 9 times. Finally, the culture was inoculated onto potato agar (PDA) medium, with 1000 μL of culture inoculated into each culture dish (11 cm in diameter), and cultured at 35° C. for 10 days. 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 a first-level strain culture, a second-level seed culture and a third-level 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 30 μL of the spore suspension inoculated on each slant. 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, the formula is: 200g peeled potatoes, 20g glucose and 15g agar, add water to 1000mL.

[0139] (2.2) Secondary seed culture

[0140] Dissolve the primary strain with 0.5% Tween 80 and then add sterile water to a concentration of 10 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 cultured 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 potatoes, 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 shake 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 culture

[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 procedure is as follows: the liquid culture is evaporated under reduced pressure at -0.08 MPa and 60°C to 1 / 10 of its original volume, dried at 130°C, and pulverized. Methanol is added at a weight-to-volume ratio of 1 g:5 mL, and ultrasonic extraction is performed at 40 kHz for 200 minutes. The extract is then centrifuged at 15,000 rpm, and the supernatant is evaporated under reduced pressure at -0.08 MPa and 60°C to remove the extractant, yielding the active ingredient 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 using 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 300 μL, column temperature is 40°C, and flow rate is 50 mL / min; detection wavelength is 358 nm; chromatographic column: preparative reverse-phase C-18 column; collecting the red eluate and the yellow eluate, evaporating the extractant under reduced pressure at a vacuum degree of -0.08 MP and a temperature of 60°C, and drying 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, namely the eluent of Monascus D, Monascus E and F. After evaporation and removal of 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, and flow rate is 20 mL / min; detection wavelength is 358 nm; chromatographic column: semi-preparative reversed-phase C-18 column.

[0155] The specific operations are as follows:

[0156] 200 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 to remove the extractant under vacuum conditions of -0.08 MP and 60° C., and dried 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 sequence to obtain the red chromatographic eluate and the red chromatographic eluate, respectively; the red chromatographic eluate and the red chromatographic eluate were respectively 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] Monascusin D is a red powder with the molecular formula C 25 H 31 NO5. 10μM Monascus D reduced the transcriptional 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. The structural formula of Monascus D is as follows:

[0159]

[0160] Monascusin E is a yellow powder with the molecular formula C 22 H 32 O5, chemically named (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 LPS-induced IL-1β, TNF-α, and TGF-β transcriptional levels in RAW264.7 cells by 67%, 59%, and 61%, respectively. This activity is similar to that of the positive control DEX, which demonstrates anti-inflammatory immune activity at the same concentration. The structural formula of red yeast rice E is as follows:

[0161]

[0162] Monascusin F is a yellow powder with the chemical name (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 transcriptional levels of IL-1β, TNF-α, and TGF-β in LPS-induced RAW264.7 cells by 77%, 86%, and 73%, respectively. This is more potent than the anti-inflammatory immune control DEX 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 A spore suspension containing 2 spores / mL was inoculated into 250 mL of PDB medium at 2% per bottle and cultured in a shake flask at 33°C and 230 r / min for 8 days. Then, 2% of this culture was inoculated into freshly prepared 230 mL of PDB medium and cultured in a shake flask at 33°C and 230 r / min for 8 days. Then, 2% of this culture was inoculated into freshly prepared 250 mL of PDB medium and cultured in a shake flask at 33°C and 230 r / min for 8 days. The previous step was then repeated, with 2% of the culture inoculated into freshly prepared 250 mL of PDB medium and cultured in a shake flask at 33°C and 230 r / min for 8 days. This inoculation culture was repeated a total of 7 times. Finally, the culture was inoculated onto potato agar (PDA) medium, with 600 μL of culture inoculated into each culture dish (11 cm in diameter), and cultured at 33° C. for 8 days. Spores were collected to obtain the domesticated red yeast rice strain RCEF7056.

[0168] (2) Preparation of solid culture

[0169] The strain of Monascus RCEF7056 was 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. The culture was carried out at a temperature of 33.5°C and a rotation speed of 235 rpm for 8 days to obtain the primary strain.

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

[0173] (2.2) Secondary seed culture

[0174] Dissolve the primary strain with 0.5% Tween 80 and then add sterile water to a concentration of 10 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 shake 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 culture

[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 procedure is as follows: the liquid culture is evaporated under reduced pressure at -0.09 MPa and 40°C to 2 / 10 of its original volume, dried at 70°C, and pulverized. An extractant is added at a weight-to-volume ratio of 1 g:2.5 mL, and ultrasonic extraction is performed at 40 kHz for 100 minutes. The extractant is then centrifuged at 10,000 rpm, and the supernatant is evaporated under reduced pressure at -0.09 MPa and 40°C to remove the extractant, yielding the active ingredient 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 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 180 μL, column temperature is 30°C, and flow rate is 25 mL / min; detection wavelength is 358 nm; chromatographic column: preparative reverse-phase C-18 column; collecting the red eluate and the yellow eluate, evaporating the extractant under reduced pressure at a vacuum degree of -0.09 MP and a temperature of 40°C, and drying at 70°C to obtain a red preliminary purified product and a yellow preliminary purified product.

[0186] (3.3) Purification of the Preliminary Purification

[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, namely, the eluent of Monascus D, Monascus E and F. After evaporation and removal of 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, and flow rate is 10 mL / min; detection wavelength is 358 nm; chromatographic column: semi-preparative reversed-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 to remove the extractant under vacuum conditions of -0.09 MP and 30° C., and dried at 60° C. to obtain 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 sequence 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 -0.09 MP and a temperature of 30°C to remove the extractant, and then dried at 60°C to obtain yellow solid red chromatographic E and yellow solid red chromatographic F, respectively.

[0192] Monascusin D is a red powder with the molecular formula C 25 H 31NO5. 10μM red yeast rice D reduced the transcriptional 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. The structural formula of red yeast rice D is as follows:

[0193]

[0194] Monascusin E is a yellow powder with the molecular formula C 22 H 32 O5, chemically named (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 LPS-induced IL-1β, TNF-α, and TGF-β transcriptional levels in RAW264.7 cells by 67%, 59%, and 61%, respectively. This activity is similar to that of the positive control DEX, which exhibits anti-inflammatory immune activity at the same concentration.

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

[0196]

[0197] Monascusin F is a yellow powder with the chemical name (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 transcriptional levels of IL-1β, TNF-α, and TGF-β in LPS-induced RAW264.7 cells by 77%, 86%, and 73%, respectively. This is more potent than the anti-inflammatory immune control DEX 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 6A spore suspension containing 2 spores / mL was inoculated into 250 mL of PDB medium at 2% per bottle and cultured in a shake flask at 34°C and 240 r / min for 9 days. Then, 2% of this culture was inoculated into a freshly prepared 240 mL of PDB medium and cultured in a shake flask at 34°C and 240 r / min for 9 days. Then, 2% of this culture was inoculated into a freshly prepared 250 mL of PDB medium and cultured in a shake flask at 34°C and 240 r / min for 9 days. The previous step was then repeated, with 2% of the culture inoculated into a freshly prepared 250 mL of PDB medium and cultured in a shake flask at 34°C and 220-250 r / min for 9 days. This inoculation culture was repeated 8 times in total. Finally, the culture was inoculated onto potato agar (PDA) medium, with 800 μL of culture inoculated into each culture dish (11 cm in diameter), and cultured at 34° C. for 9 days. 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 first-level strain culture, second-level seed culture and third-level liquid expansion culture to obtain a liquid culture;

[0205] (2.1) Primary bacterial culture

[0206] The spore suspension of Monascus rubrum was inoculated on potato agar (PDA) slant culture medium, with 20 μL of spore suspension inoculated on each slant. 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, the formula is: 200g peeled potatoes, 20g glucose and 15g agar, add water to 1000mL.

[0208] (2.2) Secondary seed culture

[0209] Dissolve the primary strain with 0.5% Tween 80 and then add sterile water to a concentration of 10 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 shake 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 culture

[0214] The specific operations are as follows:

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

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

[0217] The specific operation is as follows: the liquid culture is evaporated under reduced pressure at a vacuum of -0.1 MPa and a temperature of 10°C to 3 / 10 of its original volume, and then directly extracted with an extractant for 20 minutes. The filtrate is then filtered through a 0.1 μm membrane, and the extractant is evaporated under reduced pressure at a vacuum of -0.1 MPa and a temperature of 10°C to obtain the active ingredient extract.

[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, and flow rate is 20 mL / min; detection wavelength is 358 nm; chromatographic column: preparative reverse-phase C-18 column; collecting the red eluate and the yellow eluate, evaporating the extractant under reduced pressure at a vacuum degree of -0.1 MP and a temperature of 30°C, and drying at -40°C to obtain a red preliminary purified product and a yellow preliminary purified product.

[0221] (3.3) Purification of the Preliminary Purification

[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 evaporation and removal of the solvent, red monascus D and yellow monascus E and F were obtained.

[0223] The chromatographic separation preparation conditions 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 is 30 μL, column temperature is 30°C, flow rate is 5 mL / min; detection wavelength is 358 nm; chromatographic column: semi-preparative reversed-phase C-18 column.

[0224] The specific operations are as follows:

[0225] 150 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 to remove the extractant under vacuum conditions of -0.1 MP and 20° C., and dried at 40° C. to 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 sequence to obtain the red chromatographic eluate and the red chromatographic eluate, respectively; the red chromatographic eluate and the red chromatographic 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] Monascusin D is a red powder with the molecular formula C 25 H 31 NO5. 10μM red yeast rice D reduced the transcriptional 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. The structural formula of red yeast rice D is as follows:

[0228]

[0229] Monascusin E is a yellow powder with the molecular formula C 22 H 32 O5, chemically named (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 LPS-induced IL-1β, TNF-α, and TGF-β transcriptional levels in RAW264.7 cells by 67%, 59%, and 61%, respectively. This activity is similar to that of the positive control DEX, which exhibits anti-inflammatory immune activity at the same concentration.

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

[0231]

[0232] Monascusin F is a yellow powder with the chemical name (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 transcriptional levels of IL-1β, TNF-α, and TGF-β in LPS-induced RAW264.7 cells by 77%, 86%, and 73%, respectively. This is more potent than the anti-inflammatory immune control DEX 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 A spore suspension containing 2 spores / mL was inoculated into 250 mL of PDB medium at 2% per bottle and cultured in a shake flask at 33°C and 230 r / min for 9 days. Then, 2% of this culture was inoculated into freshly prepared 230 mL of PDB medium and cultured in a shake flask at 33°C and 230 r / min for 9 days. Then, 2% of this culture was inoculated into freshly prepared 250 mL of PDB medium and cultured in a shake flask at 33°C and 230 r / min for 9 days. The previous step was then repeated, with 2% of the culture inoculated into freshly prepared 250 mL of PDB medium and cultured in a shake flask at 32°C and 230 r / min for 9 days. This inoculation culture was repeated a total of 7 times. Finally, the culture was inoculated onto potato agar (PDA) medium, with 700 μL of culture inoculated into each culture dish (11 cm in diameter), and cultured at 33° C. for 9 days. Spores were collected to obtain the domesticated red yeast rice strain RCEF7056.

[0238] (2) Preparation of solid culture

[0239] The strain of Monascus ruber is subjected to first-level strain culture, second-level seed culture and third-level liquid expansion culture to obtain a liquid culture;

[0240] (2.1) Primary bacterial culture

[0241] The spore suspension of Monascus rubrum was inoculated on potato agar (PDA) slant culture medium, with 20 μL of spore suspension inoculated on each slant. 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, the formula is: 200g peeled potatoes, 20g glucose and 15g agar, add water to 1000mL.

[0243] (2.2) Secondary seed culture

[0244] Dissolve the primary strain with 0.5% Tween 80 and then add sterile water to a concentration of 10 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 shake 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 culture

[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 the active ingredient extract.

[0252] The specific operation is as follows: the liquid culture is evaporated under reduced pressure to 3 / 10 to 1 / 10 of its original volume under vacuum conditions of -0.1 to -0.08 MPa and a temperature of 10 to 60°C, and then directly extracted with ethyl acetate for 20 to 200 minutes. The filtrate is then filtered through a 2.5 μm membrane, and the extractant is evaporated under reduced pressure under vacuum conditions of -0.08 MPa and a temperature of 60°C to obtain an extract of the active ingredient.

[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, and flow rate is 20 mL / min; detection wavelength is 358 nm; chromatographic column: preparative reverse-phase C-18 column; collecting the red eluate and the yellow eluate, evaporating the extractant under reduced pressure at a vacuum degree of -0.1 MP and a temperature of 30°C, and drying 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 evaporation and removal of the solvent, red monascus D and yellow monascus E and F were obtained.

[0258] The chromatographic separation preparation conditions 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 is 30 μL, column temperature is 30°C, flow rate is 5 mL / min; detection wavelength is 358 nm; chromatographic column: semi-preparative reversed-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 to remove the extractant under vacuum conditions of -0.1 MP and 40° C., and dried at 80° C. to obtain red solid red chromatographic dye D.

[0261] 80 mg / mL of the red preliminary purified product was injected, and the eluates corresponding to the first highest chromatographic peak and the second highest chromatographic peak were collected in sequence to obtain the red chromatographic eluate and the red chromatographic eluate, respectively; these eluates were respectively 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 to obtain yellow solid red chromatographic E and yellow solid red chromatographic F, respectively.

[0262] Monascusin D is a red powder with the molecular formula C 25 H 31NO5. 10μM red yeast rice D reduced the transcriptional 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. The structural formula of red yeast rice D is as follows:

[0263]

[0264] Monascusin E is a yellow powder with the molecular formula C 22 H 32 O5, chemically named (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 LPS-induced IL-1β, TNF-α, and TGF-β transcriptional levels in RAW264.7 cells by 67%, 59%, and 61%, respectively. This activity is similar to that of the positive control DEX, which exhibits anti-inflammatory immune activity at the same concentration.

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

[0266]

[0267] Monascusin F is a yellow powder with the chemical name (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 transcriptional levels of IL-1β, TNF-α, and TGF-β in LPS-induced RAW264.7 cells by 77%, 86%, and 73%, respectively. This is more potent than the anti-inflammatory immune control DEX 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 scope of protection of the present invention.

Claims

1. A method for preparing red yeast rice with anti-inflammatory immune activity, characterized in that: The structural formula of red polysaccharin D is as follows: ; The structural formula of red yeast rice is as follows: ; The structural formula of red yeast rice F is as follows: ; The specific steps are as follows: (1) Strain domestication (1.1) Dissolve Monascus spores in 0.5% Tween 80 and then add sterile water to a concentration of 10 5~7 spores / mL of spore suspension was inoculated into 250 mL of PDB liquid culture medium at a rate of 1-3% of the volume of each bottle. The culture was shaken at a temperature of 32-35°C and a rotation speed of 220-250 r / min for 7-10 days. This cycle of inoculation and culture was repeated 5-9 times to obtain an acclimated liquid culture. (1.2) Inoculate the acclimated liquid culture onto potato agar medium, inoculating 200–1000 μL of the acclimated liquid culture onto each potato agar medium. Incubate at 32–35°C for 7–10 days, collect spores, and obtain liquid-cultured acclimated Monascus RCEF7056 strains. (2) Preparation and expansion of liquid culture The spore suspension of Monascus RCEF7056 was subjected to a first-level strain culture, a second-level seed culture and a third-level liquid expansion culture to obtain an expanded liquid culture; The primary bacterial culture is potato agar 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 specific extraction and purification operations are as follows: (3.1) Extraction of active ingredients from liquid culture The expanded liquid culture is concentrated under reduced pressure and dried; ultrasonic extraction is performed using an extractant, membrane filtration or centrifugal separation is performed, and the solvent is evaporated under reduced pressure to obtain an extract of an effective component; the extractant is methanol, ethyl acetate, or a mixture of methanol and ethyl acetate; (3.2) Preliminary purification of active ingredient extract 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; (3.3) Purify the two preliminary purified products separately The two preliminary purified products were purified separately by semi-preparative reversed-phase high performance liquid chromatography; The red preliminary purified product was eluted with acetonitrile solution, and the compound corresponding to the highest chromatographic peak was collected as the eluate of Monascus D. 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.

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

3. The preparation method according to claim 1, wherein: In step (3.1), the specific operations are as follows: The liquid culture is concentrated under reduced pressure to 3 / 10 to 1 / 10 of its original volume under vacuum conditions of -0.1 to -0.08 MP and a temperature of 10 to 60°C; dried at a temperature of -50 to 130°C, crushed, and an extractant is added at a weight-to-volume ratio of 1 g: 0.5 to 5 mL. Ultrasonic extraction is performed at 40 kHz for 20 to 200 minutes; the culture is 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 extractant is evaporated under reduced pressure from the filtrate or the centrifugal supernatant under vacuum conditions of -0.1 to -0.08 MP and a temperature of 10 to 60°C to obtain an active ingredient extract.

4. The preparation method according to claim 1, wherein: In step (3.2), the specific operations are as follows: Preparative reverse-phase high performance liquid chromatography was used for preliminary purification; the chromatographic separation preparation conditions were as follows: mobile phase A was ultrapure water, and mobile phase B was acetonitrile; the elution conditions were: gradient elution of 40% to 70% mobile phase B from 0 to 30 min; the injection volume was 30 to 300 μL, the column temperature was 20 to 40°C, and the flow rate was 5 to 50 mL / min; the detection wavelength was 358 nm; the chromatographic column was a preparative reverse-phase C-18 column; the red eluate and the yellow eluate were collected, and the extractant was evaporated under reduced pressure at a vacuum degree of -0.1 to -0.08 MP and a temperature of 10 to 60°C, and the products were dried at less than or equal to 130°C to obtain a red preliminary purified product and a yellow preliminary purified product.

5. The preparation method according to claim 1, wherein: In step (3.3), the specific operations are as follows: Chromatographic separation preparation conditions were as follows: mobile phase A was ultrapure water, mobile phase B was acetonitrile; elution conditions were: 40% to 70% mobile phase B from 0 to 30 min; injection volume was 20 to 200 μL, column temperature was 20 to 40°C, flow rate was 2 to 20 mL / min; detection wavelength was 358 nm; chromatographic column was a semi-preparative reversed-phase C-18 column; Inject 20-200 mg / mL of the red preliminary purified product and collect the eluate corresponding to the highest chromatographic peak to obtain a red chromatographic dye eluate. Evaporate the eluate under reduced pressure at a vacuum of -0.1-0.08 MP and a temperature of 10-60°C to remove the extractant, and dry at 130°C or less to obtain a red solid red chromatographic dye. A 20-200 mg / mL 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 sequence to obtain a red chromatographic eluate and a red chromatographic eluate, respectively. The red chromatographic eluate and the red chromatographic eluate were evaporated under reduced pressure to remove the extractant under vacuum conditions of -0.1 to -0.08 MP and a temperature of 10 to 60° C., and then dried at a temperature of less than or equal to 130° C. to obtain yellow solid red chromatographic E and yellow solid red chromatographic F, respectively.

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