CN114478565B - Monascin A with glycosidase activity inhibiting and immunoregulatory activity and preparation method thereof - Google Patents
Monascin A with glycosidase activity inhibiting and immunoregulatory activity and preparation method thereof Download PDFInfo
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- XXKNHBAFFJINCK-RVEJDSBJSA-N monascin Chemical compound C([C@@H]1[C@H](C(O[C@@]1(C)C1=O)=O)C(=O)CCCCC)C2=C1COC(\C=C\C)=C2 XXKNHBAFFJINCK-RVEJDSBJSA-N 0.000 title claims abstract description 64
- GFSMXLMQRWMHON-UHFFFAOYSA-N monascin Natural products CCCCCC(=O)C1C2C=C3C=C(OC=C3C(=O)C2(C)OC1=O)C=CC GFSMXLMQRWMHON-UHFFFAOYSA-N 0.000 title claims abstract description 64
- GIKQHOXMDCDAPT-UHFFFAOYSA-N monascusone B Natural products CC=CC1=CC2=C(CO1)C(=O)C3(C)OC(=O)C(C3C2)C(=O)C GIKQHOXMDCDAPT-UHFFFAOYSA-N 0.000 title claims abstract description 64
- 238000002360 preparation method Methods 0.000 title claims abstract description 33
- 230000002401 inhibitory effect Effects 0.000 title claims abstract description 32
- 102000005744 Glycoside Hydrolases Human genes 0.000 title claims abstract description 29
- 108010031186 Glycoside Hydrolases Proteins 0.000 title claims abstract description 29
- 230000000694 effects Effects 0.000 title claims abstract description 25
- 230000004957 immunoregulator effect Effects 0.000 title claims description 8
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical group CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims abstract description 27
- 241000228347 Monascus <ascomycete fungus> Species 0.000 claims abstract description 27
- 238000000605 extraction Methods 0.000 claims abstract description 22
- 238000012258 culturing Methods 0.000 claims abstract description 19
- 238000000034 method Methods 0.000 claims abstract description 15
- 230000002222 downregulating effect Effects 0.000 claims abstract description 9
- 238000000338 in vitro Methods 0.000 claims abstract description 9
- 230000005764 inhibitory process Effects 0.000 claims abstract description 9
- 210000002540 macrophage Anatomy 0.000 claims abstract description 9
- GOZMBJCYMQQACI-UHFFFAOYSA-N 6,7-dimethyl-3-[[methyl-[2-[methyl-[[1-[3-(trifluoromethyl)phenyl]indol-3-yl]methyl]amino]ethyl]amino]methyl]chromen-4-one;dihydrochloride Chemical compound Cl.Cl.C=1OC2=CC(C)=C(C)C=C2C(=O)C=1CN(C)CCN(C)CC(C1=CC=CC=C11)=CN1C1=CC=CC(C(F)(F)F)=C1 GOZMBJCYMQQACI-UHFFFAOYSA-N 0.000 claims abstract description 8
- 239000000843 powder Substances 0.000 claims abstract description 8
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 63
- 239000001963 growth medium Substances 0.000 claims description 42
- 238000009630 liquid culture Methods 0.000 claims description 37
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 30
- 239000000284 extract Substances 0.000 claims description 30
- 239000007788 liquid Substances 0.000 claims description 29
- 235000002595 Solanum tuberosum Nutrition 0.000 claims description 28
- 244000061456 Solanum tuberosum Species 0.000 claims description 28
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 27
- 238000001704 evaporation Methods 0.000 claims description 26
- 239000012264 purified product Substances 0.000 claims description 26
- 230000002829 reductive effect Effects 0.000 claims description 26
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 25
- 239000008103 glucose Substances 0.000 claims description 23
- 238000007670 refining Methods 0.000 claims description 23
- 238000004007 reversed phase HPLC Methods 0.000 claims description 23
- 239000004480 active ingredient Substances 0.000 claims description 21
- 239000003480 eluent Substances 0.000 claims description 21
- 238000010828 elution Methods 0.000 claims description 21
- 239000000725 suspension Substances 0.000 claims description 21
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 21
- 244000113306 Monascus purpureus Species 0.000 claims description 20
- 235000002322 Monascus purpureus Nutrition 0.000 claims description 20
- 238000001035 drying Methods 0.000 claims description 20
- 238000000746 purification Methods 0.000 claims description 18
- 150000001875 compounds Chemical class 0.000 claims description 16
- 229920001817 Agar Polymers 0.000 claims description 14
- 239000008272 agar Substances 0.000 claims description 14
- 238000001514 detection method Methods 0.000 claims description 14
- 238000002347 injection Methods 0.000 claims description 14
- 239000007924 injection Substances 0.000 claims description 14
- 235000012015 potatoes Nutrition 0.000 claims description 14
- 238000011218 seed culture Methods 0.000 claims description 14
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 14
- 239000012498 ultrapure water Substances 0.000 claims description 14
- 238000013375 chromatographic separation Methods 0.000 claims description 11
- 229940057059 monascus purpureus Drugs 0.000 claims description 10
- 239000007787 solid Substances 0.000 claims description 10
- 241000031003 Monascus ruber Species 0.000 claims description 9
- 238000012807 shake-flask culturing Methods 0.000 claims description 7
- 239000000243 solution Substances 0.000 claims description 7
- 239000008223 sterile water Substances 0.000 claims description 7
- 239000006228 supernatant Substances 0.000 claims description 7
- 239000000706 filtrate Substances 0.000 claims description 6
- 238000001914 filtration Methods 0.000 claims description 6
- 239000012528 membrane Substances 0.000 claims description 6
- 239000000203 mixture Substances 0.000 claims description 4
- 230000002519 immonomodulatory effect Effects 0.000 claims description 3
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 claims description 2
- 102000003777 Interleukin-1 beta Human genes 0.000 claims description 2
- 108090000193 Interleukin-1 beta Proteins 0.000 claims description 2
- 102100024295 Maltase-glucoamylase Human genes 0.000 claims description 2
- 102000004887 Transforming Growth Factor beta Human genes 0.000 claims description 2
- 108090001012 Transforming Growth Factor beta Proteins 0.000 claims description 2
- 108060008682 Tumor Necrosis Factor Proteins 0.000 claims description 2
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 claims description 2
- 108010028144 alpha-Glucosidases Proteins 0.000 claims description 2
- 239000008121 dextrose Substances 0.000 claims description 2
- 239000002904 solvent Substances 0.000 claims description 2
- ZRKFYGHZFMAOKI-QMGMOQQFSA-N tgfbeta Chemical compound C([C@H](NC(=O)[C@H](C(C)C)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CCSC)C(C)C)[C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O)C1=CC=C(O)C=C1 ZRKFYGHZFMAOKI-QMGMOQQFSA-N 0.000 claims description 2
- 230000003110 anti-inflammatory effect Effects 0.000 abstract description 3
- 241000233866 Fungi Species 0.000 abstract description 2
- 238000010924 continuous production Methods 0.000 abstract description 2
- 206010012601 diabetes mellitus Diseases 0.000 abstract description 2
- 238000000855 fermentation Methods 0.000 abstract description 2
- 230000004151 fermentation Effects 0.000 abstract description 2
- 230000000813 microbial effect Effects 0.000 abstract description 2
- 229940122069 Glycosidase inhibitor Drugs 0.000 abstract 1
- 239000003316 glycosidase inhibitor Substances 0.000 abstract 1
- 230000007365 immunoregulation Effects 0.000 abstract 1
- 239000003112 inhibitor Substances 0.000 abstract 1
- 230000036039 immunity Effects 0.000 description 7
- 230000001105 regulatory effect Effects 0.000 description 6
- 230000002441 reversible effect Effects 0.000 description 6
- 239000003814 drug Substances 0.000 description 5
- 238000001228 spectrum Methods 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 230000008901 benefit Effects 0.000 description 4
- 239000008280 blood Substances 0.000 description 4
- 210000004369 blood Anatomy 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 239000000047 product Substances 0.000 description 3
- 238000010298 pulverizing process Methods 0.000 description 3
- 241000228427 Eurotiales Species 0.000 description 2
- 241001095209 Monascus sp. (in: Fungi) Species 0.000 description 2
- 238000005481 NMR spectroscopy Methods 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 239000000796 flavoring agent Substances 0.000 description 2
- 235000019634 flavors Nutrition 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 230000005965 immune activity Effects 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 241000235349 Ascomycota Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 239000004215 Carbon black (E152) Substances 0.000 description 1
- 241001326554 Elaphomycetaceae Species 0.000 description 1
- 206010062016 Immunosuppression Diseases 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000012443 analytical study Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 235000021107 fermented food Nutrition 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 229930195733 hydrocarbon Natural products 0.000 description 1
- 150000002430 hydrocarbons Chemical class 0.000 description 1
- 208000026278 immune system disease Diseases 0.000 description 1
- 230000001506 immunosuppresive effect Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
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- 150000002772 monosaccharides Chemical class 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
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- 230000002195 synergetic effect Effects 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- 238000002137 ultrasound extraction Methods 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
- C07D493/02—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains two hetero rings
- C07D493/04—Ortho-condensed systems
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
- C12P17/181—Heterocyclic compounds containing oxygen atoms as the only ring heteroatoms in the condensed system, e.g. Salinomycin, Septamycin
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- General Health & Medical Sciences (AREA)
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Abstract
The invention relates to monascin A with glycosidase inhibition activity and immunoregulation activity and a preparation method thereof, belonging to the technical field of extraction and preparation of biological products. Monascin A is red powder, and molecular formula is C 25 H 32 O 7 Has effects in inhibiting glycosidase activity, and has half Inhibition Concentration (IC) on alpha-glycosidase (alpha-glycosidase) 50 ) 220 μg/mL, and has a significant downregulating effect on the IL-1β, TNF- α, and TGF- β transcript levels of in vitro cultured mouse macrophage RAW 264.7: IL-1β (0.01.+ -. 0.01), TNF-. Alpha. (0.02.+ -. 0.01), and TGF-. Beta.0.03.+ -. 0.01) were significantly altered (P < 0.05) relative to the DMSO control. The monascus A is obtained by culturing monascus and then extracting and separating; can be used for preparing glycosidase inhibitor and antiinflammatory freeEpidemic inhibitors have potential for the treatment of diabetes and immune related aspects. The monascus is edible fungi, so that the method is safe and reliable; the preparation method of the monascin A adopts microbial fermentation, is environment-friendly, is not influenced by natural environment and resources, and is easy to realize industrialized, automatic and continuous production.
Description
Technical Field
The invention belongs to the technical field of extraction and preparation of biological products, and particularly relates to extraction, purification, structure identification and activity determination of monascin A (Monascuskalin A) with glycosidase inhibition activity.
Background
Monascus sp. is a small class of filamentous saprophytic fungi belonging to the phylum Eumycota (Eumycophyta), ascomycotina (Ascomycotina), ascomycetes (Plaectomyces), eurotiales (Eurotiales), red Qu Junke (Monascaceae), monascus (Monascus), including species of Monascus purpureus (Monascus purpureus), monascus anka (Monascus anka) and Monascus ruber (Monascus ruber). Monascus can produce various enzyme substances such as saccharifying enzyme, esterifying enzyme, protease and the like, and under the synergistic effect of the enzymes, the monascus can produce flavor substances such as monosaccharide, amino acid, nucleotide and the like and aroma substances such as volatile acids, alcohols, esters and the like, and the monascus contributes significantly to the flavor of fermented foods.
In recent years, due to the improvement of living standard and the reduction of exercise quantity, the number of diabetics is increased year by year, the number of diabetics is 4.4 hundred million people currently, the number of diabetics is increased in proportion of 5% each year, the types of blood glucose reducing medicines currently are few, the choice is small, and development of new high-efficiency blood glucose reducing medicines is needed urgently. The development of related medicaments has great social and economic benefits.
The current society rapidly develops, the life and work rhythm is fast, the pressure is great, and meanwhile, the sub-health crowd suffering from immune disorder is increased year by year due to the lack of exercise and bad habits such as mobile phone watching at night, so that the development of related medicines for regulating the immunity is necessary. As a food microorganism, monascus has the advantages of safety and reliability, so that the related immune preparation and blood glucose reducing preparation have more application potential and great economic value.
Disclosure of Invention
The object of the present invention is to provide monascin a having an inhibitory activity against glycosidase and an immunomodulatory activity, and to provide a process for the preparation of monascin a.
Monascin a having glycosidase inhibitory activity and immunoregulatory activity is isolated from Monascus sp culture and has the following structural formula:
monascin A is red powder, and molecular formula is C 25 H 32 O 7 Has effects in inhibiting glycosidase activity; half maximal Inhibitory Concentration (IC) of alpha-glucosidase (alpha-glycosidase) 50 ) 220 μg/mL, and has a significant downregulating effect on the IL-1β, TNF- α, and TGF- β transcript levels of in vitro cultured mouse macrophage RAW 264.7: IL-1β (0.01.+ -. 0.01), TNF-. Alpha. (0.02.+ -. 0.01), and TGF-. Beta.0.03.+ -. 0.01) varied significantly P < 0.05 relative to the DMSO control;
the Monascus is Monascus purpureus (Monascus purpureus) or Monascus anka (Monascus anka) or Monascus ruber (Monascus ruber).
A process for preparing monascin A having glycosidase inhibitory activity and immunomodulatory activity comprises the following steps:
(1) Preparation of solid cultures
Culturing Monascus sp strain, performing primary strain culture, secondary seed culture and tertiary liquid expansion culture to obtain liquid culture;
(1.1) first-order Strain culture
Inoculating monascus suspension on potato agar (PDA) slant culture medium, inoculating 10-30 mu L of the spore suspension on each slant, culturing for 7-9 days at the temperature of 32-35 ℃ and 220-250 r/min to obtain first-class strain;
(1.2) two-stage seed culture
Preparing the first-class strain into a concentration of 10 by using sterile water 5 ～10 7 Inoculating 200-250 mu L of spore suspension of each spore/mL into 250mL of PDB culture medium, and shake-culturing for 3-5 days at the temperature of 32-35 ℃ and the rotating speed of 220-250 r/min to obtain liquid secondary strain;
(1.3) three-stage liquid expanded culture
Inoculating 1-3 mL of liquid secondary strain into 250mL of PDB culture medium, shake-flask culturing for 7-9 days at the temperature of 32-35 ℃ and the rotating speed of 220-250 r/min, and collecting the culture to obtain a liquid culture;
(2) Extraction and refinement of active principles in liquid cultures
The extractant used for extraction and refining is prepared from methanol and ethyl acetate according to the volume ratio of 0-1: 1 to 0 are evenly mixed to prepare the mixture;
the specific operation is as follows:
(2.1) extraction of active ingredient in liquid culture
Concentrating the liquid culture under reduced pressure and drying; extracting with extractant by ultrasonic wave, filtering with filter membrane or centrifuging, evaporating under reduced pressure to remove solvent to obtain effective component extract;
(2.2) preliminary purification of the extract
Performing primary purification on the active ingredient extract by adopting preparative reverse-phase high performance liquid chromatography to obtain a red primary purified product;
(2.3) refining of the preliminary purified product
Refining the primary purified product by semi-preparative reverse phase high performance liquid chromatography, and collecting the compound corresponding to the highest peak under the condition of eluting with acetonitrile solution to obtain red monascin A as monascin A eluent.
The further preparation technical requirements are as follows:
in the step (1.1), PDA culture medium, namely potato dextrose culture medium, comprises the following formula: 200g peeled potatoes, 20g glucose and 15g agar, and water was added to 1000mL.
In the steps (1.2) and (1.3), the formula of the PDB culture medium is as follows: 200g peeled potatoes, 20g glucose, and water to 1000mL.
In the step (2.1), the liquid culture is concentrated to 3/10 to 1/10 of the original volume under the conditions of vacuum degree of-0.1 to-0.08 MP and temperature of 10 to 60 ℃, dried under the conditions of temperature of-50 to 130 ℃, crushed and then 1g according to the weight-volume ratio: adding extractant into 0.5-5 mL, and extracting with 40KHz ultrasonic for 20-200 min; centrifuging or filtering with 0.1-2.5 μm membrane under 4000-15000 rpm to obtain filtrate or supernatant; the filtrate or the centrifugal supernatant is decompressed and distilled to remove the extractant under the conditions of vacuum degree of-0.1 to-0.08 MP and temperature of 10 to 60 ℃ to obtain the active ingredient extract.
In the step (2.2), refining by adopting preparative reverse-phase high performance liquid chromatography; the chromatographic separation preparation conditions are as follows: mobile phase A is ultrapure water, mobile phase B is acetonitrile; elution conditions: gradient elution is carried out on 50% -70% of mobile phase B within 0 to 30 min; the sample injection volume is 30-300 mu L, the column temperature is 20-40 ℃, and the flow rate is 5-50 mL/min; the detection wavelength is 358nm; chromatographic column: preparing a reversed phase C-18 column; collecting the eluent corresponding to the first highest peak, evaporating the extractant under reduced pressure at the vacuum degree of-0.1 to-0.08 MP and the temperature of 10-60 ℃, and drying at the temperature of less than or equal to 130 ℃ to obtain a red primary purified product.
In the step (2.3), chromatographic separation preparation conditions are as follows: mobile phase A is ultrapure water, mobile phase B is acetonitrile; elution conditions: eluting with 50-70% of mobile phase A for 0-30 min; the sample injection volume is 20-200 mu L, the column temperature is 20-40 ℃ and the flow rate is 2-20 mL/min; the detection wavelength is 358nm; chromatographic column: a semi-preparative reversed phase C-18 column, collecting the compound corresponding to the highest peak value, which is monascin A eluent; vacuum evaporating to remove extractant at 10-60 deg.C and vacuum degree of-0.1 to-0.08 MP; drying at 130 ℃ or below to obtain red solid monascin A.
In the step (2.1-2.3), the extracting agent is methanol, or ethyl acetate, or a mixture of methanol and ethyl acetate. The analytical study of the present invention is described as follows:
1. the extract was isolated and purified by preparative reverse phase high performance liquid chromatography and semi-preparative reverse phase high performance liquid chromatography to obtain 1 new compound from a Monascus sp and named monascin a.
2. Monascin a has inhibitory activity against glycosidase and half Inhibitory Concentration (IC) against alpha-glycosidase (alpha-glycosidase) 50 ) 220 μg/mL, and has a significant downregulating effect on the IL-1β, TNF- α, and TGF- β transcript levels of in vitro cultured mouse macrophage RAW 264.7: IL-1β (0.01.+ -. 0.01), TNF-. Alpha. (0.02.+ -. 0.01), and TGF-. Beta.0.03.+ -. 0.01) were significantly altered (P < 0.05) relative to the DMSO control.
4. Chemical structure identification data of monascin a
High resolution liquid chromatography-mass spectrometry analysis shows that the ionic mass-charge ratio of monascin A is 445.2224, [ M+H ]] + Corresponding molecular formula is C 25 H 32 O 7 Specific nuclear magnetic data are shown in Table 1 below.
TABLE 1 Nuclear magnetic resonance Hydrocarbon belonging Table for monascin A
The beneficial technical effects of the invention are shown in the following aspects:
1. in order to find novel natural and efficient substances with glycosidase activity inhibition and anti-inflammatory immune activity, the inventor firstly cultures a large number of Monascus strains, then extracts and activity measurement are carried out on the effective components of the culture, and a Monascus (Monascus sp.) culture extract is found to have the glycosidase activity inhibition and RAW264.7 cell inflammation inhibition and immune activity inhibition. The monascin A (Monascuskaolin A) is prepared by reverse preparation and semi-preparation chromatography separation and purification on the basis of mass culture and extraction, and the compound is red powder, and the structural formula of monascin A is obtained through one-dimensional and two-dimensional nuclear magnetic resonance and accurate mass spectrometry identification. The monascin A has stronger alpha-glycosidase inhibition activity and RAW264.7 cell immunosuppression activity, and has development potential in aspects of diabetes medicines and anti-inflammatory immunity correlation.
2. The invention discovers that Monascus sp has the function of metabolizing the active substances for the first time. On the basis of the invention, the related genes can be expected to be cloned further, and high-yield strains can be constructed.
3. As a food microorganism, monascus has the advantages of safety and reliability, so that the related immune preparation and the blood glucose reducing preparation have great application potential and economic value.
4. The invention adopts microbial fermentation production, is not influenced by natural environment and resources, is easy to realize industrialized, automatic and continuous production, and does not destroy the natural environment and the natural resources; the monascus element product produced by the process method has the advantages of low cost, simple and convenient process, stable process, easy regulation and control and high success rate.
Detailed Description
The invention is further described below with reference to examples.
Example 1
The operation steps of monascin A with the functions of inhibiting glycosidase activity and regulating immunity are as follows:
(1) Preparation of solid cultures
Culturing strain of Monascus purpureus (Monascus purpureus) by primary strain culture, secondary seed culture and tertiary liquid expansion culture to obtain liquid culture;
(1.1) first-order Strain culture
Inoculating Monascus purpureus (Monascus purpureus) spore suspension on potato agar (PDA) slant culture medium, inoculating 10 μl of spore suspension on each slant, and culturing at 32deg.C and 220r/min for 7 days to obtain primary strain.
PDA culture medium is potato glucose culture medium, and the formula is: 200g peeled potatoes, 20g glucose and 15g agar, and water was added to 1000mL.
(1.2) two-stage seed culture
Preparing the first-class strain into a concentration of 10 by using sterile water 5 200 mu L of spore suspension of each spore/mL is inoculated into 250mL of PDB culture medium at the temperature of 32 ℃ and the rotating speed of 220r/minShake flask culture for 3 days under the condition to obtain liquid secondary strain.
The formula of the PDB culture medium is as follows: 200g peeled potatoes, 20g glucose were added to 1000mL water.
(1.3) three-stage liquid expanded culture
Inoculating 1mL of liquid secondary strain into 250mL of PDB culture medium, shake-flask culturing at 32deg.C and 220r/min for 7 days, and collecting culture to obtain liquid culture.
(2) Extraction and refinement of active principles in liquid cultures
The specific operation is as follows:
(2.1) extraction of active ingredient in liquid culture
Concentrating under reduced pressure, drying, and extracting with ethyl acetate to obtain effective component extract.
The method comprises the following specific steps: evaporating the liquid culture under reduced pressure at-10deg.C and vacuum degree of-0.1 MP to 3/10 of original volume, drying at-50deg.C, and pulverizing; according to the weight-volume ratio of 1g: ethyl acetate was added at 0.5mL and extracted by 40KHz ultrasound for 20 minutes. Centrifuging at 4000 rpm, and vacuum evaporating the supernatant at-0.1 MP and 10deg.C to obtain effective component extract.
(2.2) preliminary purification of the active ingredient extract
And (3) performing primary purification on the active ingredient extract by adopting preparative reverse-phase high performance liquid chromatography to obtain a red primary purified product.
The specific operation is as follows: refining by preparative reverse phase high performance liquid chromatography; the chromatographic separation preparation conditions are as follows: mobile phase A is ultrapure water, mobile phase B is acetonitrile; elution conditions: gradient elution is carried out on the mobile phase B in a range of 50 to 70 percent within 0 to 30 minutes; sample injection volume 30 μL, column temperature 20 ℃ and flow rate 5mL/min; the detection wavelength is 358nm; chromatographic column: preparing a reversed phase C-18 column; collecting the eluent corresponding to the first highest peak, evaporating the extractant under reduced pressure at-0.1 MP and 10 deg.C, and drying at-40 deg.C to obtain red primary purified product.
(2.3) refining of the preliminary purified product
Refining the red primary purified product by semi-preparative reverse phase high performance liquid chromatography, and collecting the compound corresponding to the highest peak under the condition of eluting with acetonitrile solution to obtain monascin A eluent.
The specific operation is as follows: the chromatographic mobile phase A is ultrapure water, and the mobile phase B is acetonitrile; elution conditions: eluting with 50-70% mobile phase B for 0-30 min; sample injection volume 20. Mu.L, column temperature 20 ℃, flow rate 2mL/min; the detection wavelength is 358nm; chromatographic column: semi-preparative reverse phase C-18 column; collecting the compound corresponding to the highest peak value as monascin A eluent; evaporating under reduced pressure at 10 deg.C and vacuum degree of-0.1 MP to remove extractant; drying at-40deg.C to obtain red powder monascin A.
The structural formula of the spectrum analyzed monascin A is as follows:
monascin a has inhibitory activity against glycosidase and half Inhibitory Concentration (IC) against alpha-glycosidase (alpha-glycosidase) 50 ) 220 μg/mL, and has a significant downregulating effect on the IL-1β, TNF- α, and TGF- β transcript levels of in vitro cultured mouse macrophage RAW 264.7: IL-1β (0.01.+ -. 0.01), TNF-. Alpha. (0.02.+ -. 0.01), and TGF-. Beta.0.03.+ -. 0.01) were significantly altered (P < 0.05) relative to the DMSO control.
Example 2
The operation steps of monascin A with the functions of inhibiting glycosidase activity and regulating immunity are as follows:
(1) Preparation of solid cultures
Liquid strain of Monascus anka (Monascus anka) is subjected to primary strain culture, secondary seed culture and tertiary liquid expansion culture to obtain a liquid culture;
(1.1) first-order Strain culture
Inoculating spore suspension of Monascus anka (Monascus anka) on potato agar (PDA) slant culture medium, inoculating 30 μl of spore suspension on each slant, and culturing at 35deg.C and 250r/min for 9 days to obtain primary strain.
The PDA culture medium is potato glucose culture medium, and the formula is as follows: 200g peeled potatoes, 20g glucose and 15g agar, and water was added to 1000mL.
(1.2) two-stage seed culture
Preparing the first-class strain into a concentration of 10 by using sterile water 7 The spore suspension of each spore/mL is inoculated into 250mL of PDB culture medium according to 250 mu L of each bottle, and the culture is carried out for 5 days under the conditions of 35 ℃ and 250r/min of rotating speed by shaking bottle, thus obtaining the liquid secondary strain.
The formula of the PDB culture medium is as follows: 200g peeled potatoes, 20g glucose were added to 1000mL water.
(1.3) three-stage liquid expanded culture
Inoculating 3mL of liquid secondary strain into 250mL of PDB culture medium, shake-flask culturing at 35 ℃ and 250r/min for 9 days, and collecting culture to obtain liquid culture.
(2) Extraction and refinement of active principles in liquid cultures
The specific operation is as follows:
(2.1) extraction of active ingredient in liquid culture
Concentrating under reduced pressure, freeze drying, and extracting with methanol to obtain effective component extract.
The specific operation is as follows: evaporating the liquid culture under reduced pressure at-0.08 MP and 60 deg.C to 1/10 of original volume, drying at 130 deg.C, and pulverizing; according to the weight-volume ratio of 1g: methanol was added to 5mL and the mixture was sonicated at 40KHz for 200 min. Centrifuging at 15000 rpm, and vacuum evaporating the supernatant at-0.08 MP and 60deg.C to obtain effective component extract.
(2.2) preliminary purification of the active ingredient extract
And (3) performing primary purification on the extract by adopting preparative reverse-phase high performance liquid chromatography to obtain a red primary purified product.
The specific operation is as follows: refining by preparative reverse phase high performance liquid chromatography; the chromatographic separation preparation conditions are as follows: mobile phase A is ultrapure water, mobile phase B is acetonitrile; elution conditions: gradient elution is carried out on 50% -70% of mobile phase B within 0 to 30 min; sample injection volume 300 μL, column temperature 40 ℃ and flow rate 50mL/min; the detection wavelength is 358nm; chromatographic column: preparing a reversed phase C-18 column; collecting the eluent corresponding to the first highest peak, evaporating the extractant under reduced pressure at-0.08 MP and 60 deg.C, and drying at 130deg.C to obtain red primary purified product.
(2.3) refining of the preliminary purified product
Refining the red primary purified product by semi-preparative reverse phase high performance liquid chromatography, and collecting the compound corresponding to the highest peak under the condition of eluting with acetonitrile solution to obtain monascin A eluent.
The specific operation is as follows: the chromatographic mobile phase A is ultrapure water, and the mobile phase B is acetonitrile; elution conditions: eluting with 50-70% of mobile phase A for 0-30 min; sample injection volume 200 μL, column temperature 40 ℃ and flow rate 20mL/min; the detection wavelength is 358nm; chromatographic column: semi-preparative reverse phase C-18 column; collecting the compound corresponding to the highest peak value as monascin A eluent; evaporating under reduced pressure at 60 deg.C and vacuum degree of-0.08 MP to remove extractant; drying at 130deg.C to obtain red powder monascin A.
The structural formula of the spectrum analyzed monascin A is as follows:
monascin a has inhibitory activity against glycosidase and half Inhibitory Concentration (IC) against alpha-glycosidase (alpha-glycosidase) 50 ) 220 μg/mL, and has a significant downregulating effect on the IL-1β, TNF- α, and TGF- β transcript levels of in vitro cultured mouse macrophage RAW 264.7: IL-1β (0.01.+ -. 0.01), TNF-. Alpha. (0.02.+ -. 0.01), and TGF-. Beta.0.03.+ -. 0.01) were significantly altered (P < 0.05) relative to the DMSO control.
Example 3
The operation steps of monascin A with the functions of inhibiting glycosidase activity and regulating immunity are as follows:
(1) Preparation of solid cultures
Culturing Monascus ruber strain by primary strain culture, secondary seed culture and tertiary liquid expansion culture to obtain liquid culture;
(1.1) first-order Strain culture
Inoculating the red monascus spore suspension on potato agar (PDA) slant culture medium, inoculating 20 μl of spore suspension on each slant, and culturing at 33.5deg.C and 235r/min for 8 days to obtain first-class strain.
The PDA culture medium is potato glucose culture medium, and the formula is as follows: 200g peeled potatoes, 20g glucose and 15g agar, and water was added to 1000mL.
(1.2) two-stage seed culture
Preparing the first-class strain into a concentration of 10 by using sterile water 6 Inoculating 225 mu L of spore/mL spore suspension into 250mL of PDB culture medium, and shake-culturing at 33.5 ℃ and 240r/min for 4 days to obtain liquid secondary strain.
The formula of the PDB culture medium is as follows: 200g peeled potatoes, 20g glucose, and water to 1000mL.
(1.3) three-stage liquid expanded culture
Inoculating 2mL of liquid secondary strain into 250mL of PDB culture medium, shake-flask culturing at 33.5 ℃ and 240r/min for 8 days, and collecting culture to obtain liquid culture.
(2) Extraction and refinement of active principles in liquid cultures
The specific operation is as follows:
(2.1) extraction of active ingredient in liquid culture
Concentrating under reduced pressure, lyophilizing, and extracting with extracting agent such as methanol: ethyl acetate = 1:1 (v/v) to obtain an active ingredient extract.
The specific operation is as follows: evaporating the liquid culture under reduced pressure at 40 deg.C and vacuum degree-0.09 MP to 2/10 of original volume, drying at 70 deg.C, and pulverizing; according to the weight-volume ratio of 1g:2.5mL of the extractant is added, and the ultrasonic extraction is carried out for 100 minutes at 40 KHz. Centrifuging at 10000 rpm, and vacuum evaporating supernatant at 40 deg.C under vacuum degree of-0.09 MP to obtain effective component extract.
(2.2) preliminary purification of the active ingredient extract
And (3) performing preliminary purification on the active ingredient extract by adopting preparative reverse-phase high performance liquid chromatography to obtain a red preliminary purified product.
The specific operation is as follows: refining by preparative reverse phase high performance liquid chromatography; the chromatographic separation preparation conditions are as follows: mobile phase A is ultrapure water, mobile phase B is acetonitrile; elution conditions: gradient elution is carried out on 50% -70% of mobile phase B within 0 to 30 min; sample injection volume 180 μL, column temperature 30 ℃ and flow rate 25mL/min; the detection wavelength is 358nm; chromatographic column: preparing a reversed phase C-18 column; collecting the eluent corresponding to the first highest peak, evaporating the extractant under reduced pressure at the temperature of 40 ℃ and the vacuum degree of-0.09 MP, and drying at the temperature of 70 ℃ to obtain the red primary purified product.
(2.3) refining of the Red preliminary purification product
Refining the red primary purified product by semi-preparative reverse phase high performance liquid chromatography, and collecting the compound corresponding to the highest peak under the condition of eluting with acetonitrile solution to obtain monascin A eluent.
The specific operation is as follows: the chromatographic mobile phase A is ultrapure water, and the mobile phase B is acetonitrile; elution conditions: eluting with 50% -70% of mobile phase B for 0-30 min; sample injection volume 100. Mu.L, column temperature 30 ℃ and flow rate 10mL/min; the detection wavelength is 358nm; chromatographic column: semi-preparative reverse phase C-18 column; collecting the compound corresponding to the highest peak value as monascin A eluent; evaporating under reduced pressure at 40 deg.C and vacuum degree of-0.09 MP to remove extractant; drying at 70deg.C to obtain red powder monascin A.
The structural formula of the spectrum analyzed monascin A is as follows:
monascin a has inhibitory activity against glycosidase and half Inhibitory Concentration (IC) against alpha-glycosidase (alpha-glycosidase) 50 ) 220 μg/mL, and has transcriptional levels of IL-1 beta, TNF-alpha, and TGF-beta for in vitro culture of mouse macrophage RAW264.7Has the effect of obviously down-regulating: IL-1β (0.01.+ -. 0.01), TNF-. Alpha. (0.02.+ -. 0.01), and TGF-. Beta.0.03.+ -. 0.01) were significantly altered (P < 0.05) relative to the DMSO control.
Example 4
The operation steps of monascin A with the functions of inhibiting glycosidase activity and regulating immunity are as follows:
(1) Preparation of solid cultures
Culturing Monascus ruber strain by primary strain culture, secondary seed culture and tertiary liquid expansion culture to obtain liquid culture;
(1.1) first-order Strain culture
Inoculating the red monascus spore suspension on potato agar (PDA) slant culture medium, inoculating 20 μl of spore suspension on each slant, and culturing at 33.5deg.C at 240r/min for 8 days to obtain first-class strain.
The PDA culture medium is potato glucose culture medium, and the formula is as follows: 200g peeled potatoes, 20g glucose and 15g agar, and water was added to 1000mL.
(1.2) two-stage seed culture
Preparing the first-class strain into a concentration of 10 by using sterile water 6 Inoculating 220 mu L of spore/mL spore suspension into 250mL of PDB culture medium, and shake-culturing at 33 ℃ and 240r/min for 4 days to obtain liquid secondary strain.
The formula of the PDB culture medium is as follows: 200g peeled potatoes, 20g glucose, and water to 1000mL.
(1.3) three-stage liquid expanded culture
Inoculating 2mL of liquid secondary strain into 250mL of PDB culture medium, shake-flask culturing at 33.5 ℃ and 235r/min for 8 days, and collecting culture to obtain liquid culture.
(2) Extraction and refinement of active principles in liquid cultures
The specific operation is as follows:
(2.1) extraction of active ingredient in liquid culture
The culture was concentrated under reduced pressure, dried, homogenized and then purified with methanol: ethyl acetate = 0.5:1 (v/v) extraction to obtain an active ingredient extract.
The specific operation is as follows: evaporating the liquid culture under reduced pressure at 10 deg.C and vacuum degree-0.1 MP to 3/10 of original volume, and directly extracting with extractant for 20 min. Filtering with 0.1 μm membrane, and vacuum evaporating the filtrate under vacuum degree-0.1 MP at 10deg.C to obtain effective component extract.
(2.2) preliminary purification of the active ingredient extract is subjected to preliminary purification by preparative reverse-phase high performance liquid chromatography to obtain a red preliminary purification.
The specific operation is as follows: refining by preparative reverse phase high performance liquid chromatography; the chromatographic separation preparation conditions are as follows: mobile phase A is ultrapure water, mobile phase B is acetonitrile; elution conditions: gradient elution is carried out on 50% -70% of mobile phase B within 0 to 30 min; sample injection volume 200 μL, column temperature 30 ℃ and flow rate 20mL/min; the detection wavelength is 358nm; chromatographic column: preparing a reversed phase C-18 column; collecting the eluent corresponding to the first highest peak, evaporating the extractant under reduced pressure at-0.1 MP and 30 deg.C, and drying at-40 deg.C to obtain red primary purified product.
(2.3) refining of the preliminary purified product
Refining the red primary purified product by semi-preparative reverse phase high performance liquid chromatography, and collecting the compound corresponding to the highest peak under the condition of eluting with acetonitrile solution to obtain monascin A eluent.
The specific operation is as follows: the chromatographic separation preparation conditions are as follows: mobile phase A is ultrapure water, mobile phase B is acetonitrile; elution conditions: eluting with 50% -70% of mobile phase B for 0-30 min; sample injection volume 30 μL, column temperature 30 ℃ and flow rate 5mL/min; the detection wavelength is 358nm; chromatographic column: semi-preparative reverse phase C-18 column; collecting the compound corresponding to the highest peak value as monascin A eluent; evaporating under reduced pressure at 30 deg.C and vacuum degree of-0.1 MP to remove extractant; drying at-40deg.C to obtain red powder monascin A.
The structural formula of the spectrum analyzed monascin A is as follows:
monascin a has inhibitory activity against glycosidase and half Inhibitory Concentration (IC) against alpha-glycosidase (alpha-glycosidase) 50 ) 220 μg/mL, and has a significant downregulating effect on the IL-1β, TNF- α, and TGF- β transcript levels of in vitro cultured mouse macrophage RAW 264.7: IL-1β (0.01.+ -. 0.01), TNF-. Alpha. (0.02.+ -. 0.01), and TGF-. Beta.0.03.+ -. 0.01) were significantly altered (P < 0.05) relative to the DMSO control.
Example 5
The operation steps of monascin A with the functions of inhibiting glycosidase activity and regulating immunity are as follows:
(1) Preparation of solid cultures
Culturing Monascus ruber strain by primary strain culture, secondary seed culture and tertiary liquid expansion culture to obtain liquid culture;
(1.1) first-order Strain culture
Inoculating the red monascus spore suspension on potato agar (PDA) slant culture medium, inoculating 20 μl of spore suspension on each slant, and culturing at 33.5deg.C at 240r/min for 8 days to obtain first-class strain.
The PDA culture medium is potato glucose culture medium, and the formula is as follows: 200g peeled potatoes, 20g glucose and 15g agar, and water was added to 1000mL.
(1.2) two-stage seed culture
Preparing the first-class strain into a concentration of 10 by using sterile water 6 Inoculating 220 mu L of spore/mL spore suspension into 250mL of PDB culture medium, and shake-culturing at 33 ℃ and 240r/min for 4 days to obtain liquid secondary strain.
The formula of the PDB culture medium is as follows: 200g peeled potatoes, 20g glucose, and water to 1000mL.
(1.3) three-stage liquid expanded culture
2mL of liquid secondary bacteria are inoculated into 250mL of PDB culture medium, shake flask culture is carried out for 8 days under the conditions of the temperature of 33.5 ℃ and the rotating speed of 235r/min, and the culture is collected, thus obtaining the liquid culture.
(2) Extraction and refinement of active principles in liquid cultures
The specific operation is as follows:
(2.1) extraction of active ingredient in liquid culture
The culture was concentrated under reduced pressure and then homogenized with methanol: ethyl acetate = 1:0.5 (v/v) extraction to obtain an active ingredient extract.
The specific operation is as follows: evaporating the liquid culture under reduced pressure to 3/10-1/10 of the original volume under the conditions of vacuum degree of-0.1 to-0.08 MP and temperature of 10-60 ℃, and directly extracting with ethyl acetate for 20-200 minutes. Filtering with 2.5 μm membrane, and vacuum evaporating the filtrate under vacuum degree-0.08 MP at 60deg.C to obtain effective component extract.
(2.2) preliminary purification of the active ingredient extract
And (3) performing primary purification on the extract by adopting preparative reverse-phase high performance liquid chromatography to obtain a red primary purified product.
The specific operation is as follows: refining by preparative reverse phase high performance liquid chromatography; the chromatographic separation preparation conditions are as follows: mobile phase A is ultrapure water, mobile phase B is acetonitrile; elution conditions: gradient elution is carried out on 50% -70% of mobile phase B within 0 to 30 min; sample injection volume 200 μL, column temperature 30 ℃ and flow rate 20mL/min; the detection wavelength is 358nm; chromatographic column: preparing a reversed phase C-18 column; collecting the eluent corresponding to the first highest peak, evaporating the extractant under reduced pressure at-0.1 MP and 30 deg.C, and drying at-40 deg.C to obtain red primary purified product.
(2.3) refining of the preliminary purified product
Refining the red primary purified product by semi-preparative reverse phase high performance liquid chromatography, and collecting the compound corresponding to the highest peak under the condition of eluting with acetonitrile solution to obtain monascin A eluent.
The specific operation is as follows: the chromatographic separation preparation conditions are as follows: mobile phase A is ultrapure water, mobile phase B is acetonitrile; elution conditions: eluting with 50% -70% of mobile phase B for 0-30 min; sample injection volume 30 μL, column temperature 30 ℃ and flow rate 5mL/min; the detection wavelength is 358nm; chromatographic column: semi-preparative reverse phase C-18 column; collecting the compound corresponding to the highest peak value as monascin A eluent; evaporating under reduced pressure at 30 deg.C and vacuum degree of-0.1 MP to remove extractant; drying at-40deg.C to obtain red solid monascin A.
The structural formula of the spectrum analyzed monascin A is as follows:
monascin a has inhibitory activity against glycosidase and half Inhibitory Concentration (IC) against alpha-glycosidase (alpha-glycosidase) 50 ) 220 μg/mL, and has a significant downregulating effect on the IL-1β, TNF- α, and TGF- β transcript levels of in vitro cultured mouse macrophage RAW 264.7: IL-1β (0.01.+ -. 0.01), TNF-. Alpha. (0.02.+ -. 0.01), and TGF-. Beta.0.03.+ -. 0.01) were significantly altered (P < 0.05) relative to the DMSO control.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.
Claims (5)
1. Monascin a having glycosidase inhibitory activity and immunomodulatory activity, characterized in that: the Monascus A is isolated and prepared from Monascus sp culture, and has the following structural formula:
monascin A is red powder, and molecular formula is C 25 H 32 O 7 Has effects in inhibiting glycosidase activity; half maximal Inhibitory Concentration (IC) of alpha-glucosidase (alpha-glycosidase) 50 ) 220 μg/mL, and has a significant downregulating effect on the IL-1β, TNF- α, and TGF- β transcript levels of in vitro cultured mouse macrophage RAW 264.7: IL-1 beta (0.01.+ -. 0.01), TNF-alpha (0.02.+ -. 0.01), and TGF-beta (0.03.+ -. 0.01) were significantly P </relative to DMSO control changes0.05。
2. The method for preparing monascin a with glycosidase inhibitory activity and immunoregulatory activity according to claim 1, wherein the method comprises the following steps: the Monascus is Monascus purpureus (Monascus purpureus) or Monascus anka (Monascus anka) or Monascus ruber (Monascus ruber);
the preparation method of monascin A with glycosidase inhibition activity and immunoregulatory activity comprises the following steps:
(1) Preparation of solid cultures
Culturing Monascus sp strain, performing primary strain culture, secondary seed culture and tertiary liquid expansion culture to obtain liquid culture;
(1.1) first-order Strain culture
Inoculating monascus suspension on potato agar PDA slant culture medium, inoculating 10-30 mu L of the spore suspension on each slant, culturing for 7-9 days at the temperature of 32-35 ℃ and 220-250 r/min to obtain first-class strain;
the formula of PDA slant culture medium, namely potato dextrose culture medium, is as follows: 200g peeled potatoes, 20g glucose and 15g agar, and adding water to 1000mL;
(1.2) two-stage seed culture
Preparing the first-class strain into a concentration of 10 by using sterile water 5 ～10 7 Inoculating 200-250 mu L of spore suspension of each spore/mL into 250mL of PDB culture medium, and shake-culturing for 3-5 days at the temperature of 32-35 ℃ and the rotating speed of 220-250 r/min to obtain liquid secondary strain;
the formula of the PDB culture medium is as follows: 200g of peeled potatoes and 20g of glucose, and adding water to 1000mL;
(1.3) three-stage liquid expanded culture
Inoculating 1-3 mL of liquid secondary strain into 250mL of PDB culture medium, shake-flask culturing for 7-9 days at the temperature of 32-35 ℃ and the rotating speed of 220-250 r/min, and collecting the culture to obtain a liquid culture;
(2) Extraction and refinement of active principles in liquid cultures
The extractant used for extraction and refining is prepared from methanol and ethyl acetate according to the volume ratio of 0-1: 1 to 0 are evenly mixed to prepare the mixture;
the specific operation is as follows:
(2.1) extraction of active ingredient in liquid culture
Concentrating the liquid culture under reduced pressure and drying; extracting with extractant by ultrasonic wave, filtering with filter membrane or centrifuging, evaporating under reduced pressure to remove solvent to obtain effective component extract;
(2.2) preliminary purification of the extract
Performing primary purification on the active ingredient extract by adopting preparative reverse-phase high performance liquid chromatography to obtain a red primary purified product;
(2.3) refining of the preliminary purified product
Refining the primary purified product by semi-preparative reverse phase high performance liquid chromatography, and collecting the compound corresponding to the highest peak under the condition of eluting with acetonitrile solution to obtain red monascin A as monascin A eluent.
3. The method for preparing monascin a with glycosidase inhibitory activity and immunoregulatory activity according to claim 2, wherein the method comprises the following steps: in the step (2.1), the liquid culture is concentrated to 3/10 to 1/10 of the original volume under the conditions of vacuum degree of-0.1 to-0.08 MPa and temperature of 10 to 60 ℃, dried and crushed under the conditions of temperature of-50 to 130 ℃ and the weight-volume ratio of 1g: adding extractant into 0.5-5 mL, and extracting with 40KHz ultrasonic for 20-200 min; centrifuging or filtering with 0.1-2.5 μm membrane under 4000-15000 rpm to obtain filtrate or supernatant; the filtrate or the centrifugal supernatant is decompressed and distilled to remove the extractant under the condition of vacuum degree of-0.1 to-0.08 MPa and temperature of 10-60 ℃ to obtain the active ingredient extract.
4. The method for preparing monascin a with glycosidase inhibitory activity and immunoregulatory activity according to claim 2, wherein the method comprises the following steps: in the step (2.2), refining by adopting preparative reverse-phase high performance liquid chromatography; the chromatographic separation preparation conditions are as follows: mobile phase A is ultrapure water, mobile phase B is acetonitrile; elution conditions: gradient elution is carried out on 50% -70% of mobile phase B within 0 to 30 min; the sample injection volume is 30-300 mu L, the column temperature is 20-40 ℃, and the flow rate is 5-50 mL/min; the detection wavelength is 358nm; chromatographic column: preparing a reversed phase C-18 column; collecting the eluent corresponding to the first highest peak, evaporating the extractant under reduced pressure at the vacuum degree of-0.1 to-0.08 MPa and the temperature of 10-60 ℃, and drying at the temperature of less than or equal to 130 ℃ to obtain a red primary purified product.
5. The method for preparing monascin a with glycosidase inhibitory activity and immunoregulatory activity according to claim 2, wherein the method comprises the following steps: in the step (2.3), chromatographic separation preparation conditions are as follows: mobile phase A is ultrapure water, mobile phase B is acetonitrile; elution conditions: eluting with 50-70% of mobile phase A for 0-30 min; the sample injection volume is 20-200 mu L, the column temperature is 20-40 ℃ and the flow rate is 2-20 mL/min; the detection wavelength is 358nm; chromatographic column: a semi-preparative reversed phase C-18 column, collecting the compound corresponding to the highest peak value, which is monascin A eluent; vacuum evaporating to remove extractant under vacuum degree of-0.1 to-0.08 MPa and temperature of 10-60 ℃; drying at 130 ℃ or below to obtain red solid monascin A.
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| CN115611914A (en) * | 2022-10-20 | 2023-01-17 | 安徽农业大学 | Monascus A, B and C with glycosidase inhibiting activity, immunoregulation activity and anti-aging activity and preparation method thereof |
| CN115650944B (en) * | 2022-11-10 | 2024-02-02 | 华中农业大学 | Uric acid-reducing compound |
| CN117298177B (en) * | 2023-11-28 | 2024-02-02 | 暨南大学 | Natural immunomodulator and preparation method thereof |
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